Method of suppressing immune responses by reducing intracellular content of glutathione in macrophages and monocytes

ABSTRACT

A method of suppressing immune responses, comprising administering to a patient in need thereof an effective amount of a composition comprising a substance capable of reducing the content of reductive glutathione in macrophages.

This application is a Continuation-in-Part of U.S. application Ser. No.09/181,881, filed Oct. 29, 1998, pending.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to a novel immunomodulator. Morespecifically, the present invention relates to an immunomodulator (e.g.,immunoenhancer and immunosuppressant) capable of oral intake which has anovel suppressive function on macrophages (hereinafter sometimesabbreviated as “MΦ” ) or monocytes and which may be used, for example,for the treatment, improvement and prevention of human immunologicaldiseases such as hepatic cirrhosis, hepatitis, diabetes,gastrointestinal inflammatory diseases such as inflammatory boweldiseases (ulcerative colitis, Crohn disease, etc.), auto-immune diseasesand allergic diseases such as hypersensitive interstitial pneumonia,pulmonary fibrosis, chronic rheumatoid arthritis, asthma and cutaneousatopy, and cancers, and to a drug, a food (including a food for medicalcare, a health food or a special sanitary food), a nutrient and aninfusion containing the same.

2. Description of the Related Art

As used herein, the term immune system refers to a system in an organismfor defending itself from exogenous infection with virus, bacteria orthe like, or from invasion of a human body with transformed cells (tumorcells and the like) formed by transformation of autologous cells.However, the immune system occasionally behaves abnormally, i.e., itfunctions excessively and acts to reject autologous components, or, onthe other hand it sometimes functions deficiently, resulting in animmunocompromised state. Diseases resulting from these abnormalresponses are generally called immunological diseases. Examples thereofinclude diverse diseases, for example, acute or chronic inflammatorydiseases such as atopic cutaneous inflammatory diseases, pollinosis,asthma and sarcoidosis; autoimmune diseases such as allergic diseases,chronic rheumatoid arthritis, diabetes (IDDM), SLE and chronic fatiguesyndrome; hepatitis, hepatic cirrhosis, inflammatory bowel diseases(IBD) such as ulcerative colitis and Crohn disease; and cancer cachexia.These immunological diseases originate from complex pathological causes.Systemic immunodeficiency and functional deficiency originate frompathological inflammation accompanied by cell proliferation,differentiation or cell necrosis through local production of cytokinesor inflammatory mediators.

As cells that participate in immunity, T lymphocytes and B lymphocytesare well known. These cells exhibit a wide variety of functions as cellsplaying roles in cellular immunity and humoral immunity respectively.Meanwhile, macrophages and monocytes are cells that are intimatelyinvolved in both cellular immunity and humoral immunity, and theyparticipate significantly in rejection of non-self foreign bodies, forexample, in immunological diseases such as allergy and rheumatism,cancers and bacterial infection.

The functions of macrophages and monocytes are classified into fourdifferent types, a secretary function, an immunoregulatory function(mainly antigen presentation), treatment of foreign bodies and wastematters, a phagocytic function and a cytotoxic/cytostatic activityagainst target cells. It is widely accepted that these cells producediverse inflammatory mediators; for example cytokines such as TNF,IL-12, IL-1, IL-6, TGFβ and IL-8 and so on; hormonal molecules such asneopterine (NPT) and dihydroxyepiandrosterone (DHEA); arachidonic acidmetabolites such as PGE2 and LTB4; complement and related molecules suchas C5a and C3; such as reactive oxygen and reactive nitrogenintermediates. It has not been clarified whether these diverse functionsare exhibited by one kind of macrophage or monocyte or by distinctivegroups of macrophages or monocytes having different functions. Whilelymphocytes are classified into distinctive subsets according to theircell surface markers and the distinctive functional markers uniquelycorrespond to each subset of lymphocyte, the correspondence between thewide variety of functions of macrophages is less clear. Monocytes havenot been classified into cellular subsets. For this reason, althoughmacrophages and monocytes play quite important roles in the triggeringand the pathological progression of the above mentioned inflammatory,allergic and immunological diseases, the functional classification ofmacrophages and monocytes subsets has not yet been applied at all totherapeutic, prophylactic and preventive treatment of human diseases,with the assumption of the presence of macrophage and monocyte subsets,and even the hypothesis thereof has not yet been given.

In recent years, in the patients suffering from allergic diseases,autoimmune-diseases such as chronic rheumatoid arthritis and cancer, theinclination of helper T cell subsets in the peripheral blood has beenpointed out and has been linked to the pathology of these diseases.Helper T lymphocytes which are a subset of T lymphocytes have beenfurther classified into two subsets, namely Th1 and Th2, and it iscurrently proving that the ratio of these two types is an relevant indexof immunological functions of patients. Attempts are being made toestablish a more appropriate therapeutic treatment by diagnosis of theratio or by improvement of the ratio based on this index. That is, it isknown that when the amount of Th2 inducing IgE production from B cellsis higher than that of Th1 (Th1<Th2), allergic diseases are worsened.Attempts are being made to suppress allergy upon measuring a Th1/Th2ratio to examine an immunological response of patients or to provide Th1response superior to Th2 responses. On the contrary, the presence ofdiseases caused by a predominance of Th1 has been successively indicatedalso in chronic rheumatoid arthritis or an asthmatic inflammatorydisease at the chronic stage.

Even when the Th1/Th2 balance is measured using biological materials andthe functions of the two subsets are modulated, this modulation has notsuccessfully been utilized currently in the examination or the diagnosisof local chronic inflammatory diseases or allergic diseases. The termssuch as Th1 diseases and Th2 diseases have been used lately. However,these terms cannot necessarily be distinguished clearly.

The Th1/Th2 presence ratio is only an index of lymphocyte subsets. Sincethe in vivo dynamism of the lymphocyte subsets is actually regulated bythe cell group called accessory cells including macrophages in thepresent invention, it is difficult to appropriately diagnose theprogression of diseases with only the Th1/Th2 presence ratio and totreat the same on the basis of this index. As will be described below,the Th1/Th2 balance is controlled by the distinct macrophage/monocytesfunctions. Even if a skewing to Th1>Th2 is intended, this is hardlyeffective for therapy of immunological diseases, due to the presence ofa complex cytokine network, and a new index for diagnosis and therapyhas been in demand.

It has been clarified that in macrophages participating in theinflammatory reactions, the functions of the cells are variabledepending on environmental factors such as oxidative stress, cytokinestimulation, infection with virus or bacteria and the like. However, thecorrespondence between the functions and the classification of cellsubsets of macrophages is highly uncertain. New findings are required inthe above-mentioned classification of functions and subsets, and thesefindings will lead to the development of quite useful new therapeuticmethods. Under such circumstances, the development of excellent agentsfor modulating immunity, namely, immunomodulators, has been in demand.

SUMMARY OF THE INVENTION

It is an object of the present invention to provide a method ofsuppressing immune responses.

It is another object of the present invention to provide compositions,especially food compositions, which may be used to suppress immuneresponses.

The present inventors have conducted investigations to solve theabove-mentioned problems, and have consequently found the followingfindings. That is, they have attempted to distinguish macrophages(including monocytes) which share with an immunosuppressive activity, acachexia inducing activity, an activity of inducing malignantprogression and an activity of prolonging inflammation fromimmunoregulatory macrophages in view of a difference in a redox state(potential) of macrophages, and have then succeeded in this attempt. Thereductive glutathione (GSH) content in macrophages is employed as anindex thereof.

Accordingly, the objects of the present invention, and others, may beaccomplished with method of suppressing immune responses byadministering to a patient in need thereof an effective amount of acomposition comprising a substance capable of reducing the content ofreductive glutathione in macrophages.

BRIEF DESCRIPTION OF THE DRAWINGS

A more complete appreciation of the invention and many of the attendantadvantages thereof will be readily obtained as the same becomes betterunderstood by to the following detailed description when considered oncombination with the accompanying drawings, wherein:

FIG. 1 is a diagrammatic view showing a relationship of a difference ina function of macrophages, with respect to the Th1 and Th2 balance,immunosuppression, malignant progression, cancer cachexia, and localinflammatory responses.

FIG. 2 illustrates that the presence ratio of oxidative and reductivemacrophages controls the immunological functions through the skewedgeneration of Th1 and Th2 cytokines. This is based on the new findingsof the present inventors, showing that the redox condition ofmacrophages plays an important role in amplifying the inclination of thein vivo responses between humoral and cellular immunity.

FIG. 3 is a view showing the results of the examination of functions ofboth macrophages, namely the functional differences between oxidativemacrophages and reductive macrophages.

FIG. 4 is a view showing the results of examining whether there is adifference in the IL-12 production between Lentinan (LNT) induced MΦ andLipopolysaccharide (LPS) induced MΦ. It indicates that there is a greatdifference in the amount of IL-12 (Th1 inducing cytokine) producedbetween oxidative and reductive macrophages and IL-12 is produced onlyfrom reductive macrophages with the high intracellular reductiveglutathione content.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Glutathione is present in all mammalian cells, and well known as anintrinsic antioxidant. It is a tripeptide having a wide variety offunctions in cells, such as removal of radicals or peroxides, metabolismof eicosanoids such as prostaglandin, detoxication of biologicallyforeign materials, amino acid transport and the like. Glutathioneincludes reductive glutathione (GSH) and oxidative glutathione (GSSG),and these form a coupled cycle. In normal cells, the content ofreductive glutathione (GSH) is higher, and it acts defensively onoxidative stress, especially on H₂O₂.

Ruede et al. have already reported that with respect to macrophagesdifferentiated in the presence of GM-CSF and macrophages differentiatedin the presence of M-CSF from monocytes, the cellular GSH content of theformer is higher than that of the latter, so that the difference in theGSH content in cells seems likely to participate in the function ofmacrophages (Germann, T., Mattner, F., Partenheimer, A. et al.:Different accessory function for Th1 cells of bone marrow derivedmacrophages cultured in granulocyte-macrophage colony-stimulating factoror macrophage colony-stimulating factor. Int. Immunol., 4:755, 1992:Frosch, S., Bonifas, U., Eck, H. P. et al. : The efficient bovineinsulin presentation capacity of bone marrow-derived macrophagesactivated by granulocyte-macrophage colony-stimulating factor correlateswith a high level of intracellular reducing thiols. Eur. J. Immunol.,23; 430, 1993). The present inventors have measured the reductive GSHcontent in macrophages, and have found that there is a great differencein an immunological function between macrophages having different GSHcontents (refer to FIG. 1); they have tested the immune responses withregards to the cellular GSH content, and have found that the redoxstates can artificially be modulated with an orally administerablelow-molecular weight substance, and that these substances capable tomodulate intracellular GSH content can widely be applied to treatment ofwide variety of diseases and the substance can be also used as a food(refer to FIG. 1). These findings have led to the completion of thepresent invention.

FIG. 1 is a diagrammatic view showing a linkage between a difference ina function of macrophages or monocytes (both are referred to as“macrophages” in the present invention), and an effect on a Th1/Th2balance, a mechanism of immunosuppression, cachexia induction andinduction of malignant tumor progression caused by a functionaldifference of macrophages and local inflammatory diseases. For example,according to the tumor progression, the local Th1/Th2 balance is skewed,an inclination to humoral immunity appears, the structure and thefunction of the cytokine receptor complex are changed, oxidativemacrophages with a low intracellular GSH content are increased, theproduction of active oxygen or inflammatory mediators such as PGE2,IL-6, IL-10 and IL-8 are increased to cause systemic immunosuppressionor induction of cachexia and to prolong chronic inflammation accompaniedby allergic reactions or tissue injury.

The present inventors have conducted further investigations on the basisof the above-mentioned findings, and have consequently found thatheterogeneous macrophages, which play important roles in theinflammation reactions can be classified into two groups, namely,oxidative macrophages and reductive macrophages by determining theintracellular content of oxidative glutathione and the intracellularcontent of reductive glutathione in macrophages. The oxidativemacrophages induce local chronic inflammatory diseases or an allergicreaction in immunological diseases and the Th1/Th2 balance controllingthe balance of humoral and cellular immunity is regulated with the redoxstate of macrophages, that the redox state of the macrophages plays animportant role in immunological diseases, and this redox state ismonitored and artificially controlled or modified which is useful in thediagnosis or the therapy of these immunological diseases, and that thiscontrol can easily be conducted using low-molecular weight substancescapable of oral intake.

With respect to the definition of the oxidative macrophage and thereductive macrophage in the present invention, macrophages are reactedwith monochlorobimane which is a chemical reagent specific to reductiveglutathione (GSH) to determine the GSH content in cells. A macrophagehaving a GSH content which is higher in comparison with the residentmacrophages is defined as a reductive macrophage, and a macrophagehaving a GSH content which is lower is defined as an oxidativemacrophage. Further, a macrophage in which the GSH content becomes morethan 2 nmoles/5×10⁵ macrophages by bringing a low-molecular weightsubstance capable of oral intake into contact with the macrophage forfrom 2 to 24 hours is defined as the reductive macrophage (or monocyte),and a macrophage in which the GSH content becomes less than 0.1nmoles/5×10⁵ macrophages is defined as the oxidative macrophage.Alternatively, a macrophage of which the GSH content is two or moretimes than that of the resident macrophage is defined as a reductivemacrophage, and a macrophage of which the GSH content is ⅕ or less thatof the resident macrophage is defined as an oxidative macrophage.

At present, it is considered that the Th1/Th2 balance is regulated bythe ratio of IL-6 or IL-4 and IL-12 produced in vivo. It is known thatTh2 participating in humoral immunity is induced by the former two andTh1 by IL-12 respectively. It is clarified that IL-6 and IL-12 areproduced from macrophages. However, assuming that the same macrophagesproduce both IL-6 and IL-12, one type of a macrophage participating inboth the Th1 induction and the Th2 induction comes to be present. Thus,there is a great contradiction in considering the host immune responses.

The present inventors have found that IL-12 is produced from only thereductive macrophage having the high intracellular GSH content to act onthe Th1 induction and that the IL-6 production is increased in theoxidative macrophage to induce Th2. They have further found that whenthe macrophage is inclined to the oxidative type in spite of theproduction of IFNγ, a typical Th1 cytokine, IL-6 skewing the balance toTh2 is produced in a large amount by IFNγ stimulation. On the contrary,it has also been found that IFNγ, a typical Th1 cytokine, increases thephenotype of the reductive macrophage further by acting on the reductivemacrophage. When IL-4, the typical Th2 cytokine, acts on the oxidativemacrophage, the oxidative macrophage phenotypes are further increased.These knowledge indicate that the balance between humoral immunity andcellular immunity is unequivocally defined by the redox state ofmacrophages, and they are relevant new findings innovating the basicconcept of immunology (refer to FIG. 2). On the basis of these findings,the quite useful, original invention overcoming the conventionalconfused immunological disease therapy was already completed withrespect to the diagnosis and the therapy of the immunological diseases.Investigations have been assiduously conducted on the basis of theabove-mentioned findings. Consequently, the present invention has beennewly completed.

That is, the present invention is an immunomodulator containingsubstances having an activity of changing a content of glutathione inmacrophages. In the present invention, the macrophage also includesmonocytes. This substance is preferably one which provide MΦ withproductivity of interleukin 12 by increasing the content of reductiveglutathione in macrophages. More preferable examples thereof includelow-molecular weight substances, for example, a GSH precursormetabolized into GSH within cells, such as N-acetylcysteine (NAC),γ-glutamylcysteine, γ-glutamylcysteine dimethyl ester,γ-glutamylcysteine diethyl ester and N-acetylcysteine nitroxybutylester; glutathione derivatives such as glutathione monoester,glutathione diester and glutathione nitroxybutyl ester; lipoic acid andderivatives thereof; gliotoxin and derivatives thereof having two SHgroups or more in the molecule; and ortene. These can be administeredorally or percutaneously. It is also possible to use antioxidants suchas flavonoid and derivatives thereof which raise the GSH content,increase the production of IL-12 and decrease the production of IL-6 bycontact with macrophages. Further, high-molecular weight substanceswhich are used in combination therewith, such as β(1-3) glucan andcytokine, are preferably used in the intravenous administration and theadministration using DDS (drug delivery system). Preferable examples ofthe cytokine include IL-4, IL-2, IL-12, TGFβ and IFNγ. When it isrequired to increase cellular immunity, IL-2 and/or IFNγ is especiallypreferable. When it is required to decrease cellular immunity, IL-4and/or TGFβ is especially preferable. These substances can be containedeither solely or in combination, and a higher effect is expected by acombination of a low-molecular weight orally administerableimmunomodulator and a high-molecular weight immunomodulator suited forintravenous administration.

Further, the present invention also includes an immunomodulatorcontaining a substance which can selectively remove either of two typesof macrophages, reductive macrophages or oxidative macrophages which aredifferent in the intracellular content of reductive glutathione.Examples of the substance include a substance in which a cytotoxic DNAalkylating agent is conjugated with glutathione, and a substance inwhich an oxidative or reductive macrophage-specific antibody isconjugated directly or through a linker with a low-molecular weightcompound having a cytotoxicity to macrophages or with a material showinga cytotoxicity after being incorporated into a macrophage. Examples ofthe alkylating agent include cyclophosphamide, nimustine (ACNU),mitomycin C and melphalan. In an oxidative macrophage in whichglutathione S-transferase is activated, an DNA alkylating agent, boundto glutathione directly or through a linker, is deconjugated by theaction of this enzyme and can remove the reductive macrophage byspecifically killing the same. Further, a substance which has nocell-killing property in vitro but comes to show the cell-killingproperty with the action of an enzyme increased either in oxidative orreductive macrophages can also be used as a prodrug.

The present inventors have conducted further investigations especiallyon gastrointestinal inflammatory diseases based on these findings, havelooked for substances of reducing a content of reductive glutathione inmacrophages, and have found substances which exhibit the effect at quitelow doses. They have produced animal models having gastrointestinalinflammatory diseases similar to those of humans, and have promotedtheir investigations for development of an immunosuppressant as a drug,and a food such as a food for medical care; a health food or a specialsanitary food, a nutrient and an infusion which have animmunosuppressive activity. They have focused on cystine derivatives,and have widely examined an effect of inhibiting an immunologicalactivity in vitro and an immunosuppressive effect in the administrationto animals using a compound represented by the following structuralformula (1), as well as a pharmaceutical effect of candidate substancesusing gene knock out mice which are spontaneously accompanied withgastrointestinal inflammatory diseases. Consequently, they have foundthat cystine derivatives, especially those represented by the followingstructural formula (1) are effective as pharmaceutical substances havingan activity of reducing the content of reductive glutathione inmacrophages and monocytes.

wherein R¹ and R², independently from each other, represent an alkylgroup or a substituted alkyl group such as the nitroxybutyl group, andR³ and R⁴, independently from each other, represent an acyl group or apeptidyl group.

Especially, it is quite useful as an immunosuppressant againstinflammatory bowel diseases such as ulcerative colitis and Crohndisease, and gastrointestinal inflammatory diseases such as hepatitisand hepatic cirrhosis.

Further, the present invention includes a food (including a food formedical care, a health food or a special sanitary food), a nutrient oran infusion containing the above-mentioned immunomodulator. The foodincludes ordinary foods and those which are put into the mouth, such asa toothpaste, a chewing gum and the like. It is especially preferable toincorporate the immunomodulator in health foods. Further, it may be usedas an additive which is added to a food. As the nutrient, vitaminpreparations and calcium preparations are available. As the infusion, ahigh calorie infusion, a physiological saline solution and bloodpreparations are available.

In addition, the immunomodulator, the food, the nutrient and theinfusion of the present invention is preferably used for improvement ofthe cachectic condition of patients suffering from cancers and fordiabetes, gastrointestinal inflammatory diseases, chronic rheumatoidarthritis, hepatitis, hepatic cirrhosis, above stated autoimmuneinflammatory diseases and/or chemoprevention of cancers.

Especially, an immunomodulator having an activity of reducing a contentof reductive glutathione in macrophages of humans can widely be used innot only drugs but also the foods as an immunosuppressant of theautoimmune inflammatory diseases in the form of a single compound or amixture. Thus, it is useful as the food, the nutrient and the infusionhaving the immunosuppressive activity.

The present invention is described in even more detail below.

The present invention provides an immunomodulator useful for therapy ofpatients suffering from immunological diseases in which macrophages areclassified into oxidative macrophages and reductive macrophages havingdifferent functions by determining the contents of oxidative glutathioneand/or reductive glutathione in macrophages using a body fluid or a cellsample separated and collected from humans, and the ratio of thesemacrophages present is artificially controlled with a substance capableof oral intake or either oxidative macrophages or reductive macrophagesare artificially removed, as well as to a food, a nutrient and aninfusion which are useful for improvement of diseases. With respect tothe body fluid/cell samples separated and collected from humans, thereare, for example, cells separated from the peripheral blood, theperitoneal cavity, the thoracic cavity, the solid carcinoma localtissue, the articular cavity and various organs.

With respect to a method of measuring a content of glutathione, acontent of oxidative or reductive glutathione is directly measuredbiochemically by an enzymatic recycling method [refer to Protocol ofActive Enzyme Experiments (Saibo Kogaku, separate volume), Shujunsha,pp. 84-88, 1994, Analytical Chemistry, vol. 106, pp. 207-212, 1980, andCellular Immunology, vol. 164, pp. 73-80, 1995]. Each of thesepublications is incorporated herein by reference. Further, it canindirectly be measured using a monoclonal antibody or a polyclonalantibody specific to oxidative or reductive macrophages or using areagent which is specifically reacted with GSH to form a complex andemits fluorescent light through laser excitation, such asmonochlorobimane.

Glutathione in the present invention is also calledL-glutamyl-L-cysteinylglycine. It is an SH compound which is mostlypresent in vivo, and generally referred to as “GSH”. Glutathione isclassified into reductive glutathione and oxidative glutathione.Reductive glutathione refers to the above-mentioned glutathione (GSH).Oxidative glutathione is also called glutathione disulfide, and isreferred to as “GSSG”.

The macrophage in the present invention also includes theabove-mentioned monocyte. Macrophage-related cells called dendriticcells and Kupffer cells are also included. The macrophage is known tosecrete or release various mediators such as cytokines and inflammatorymediators from cells thereof. Whether they are secreted or not isdetermined depending on its activated or differentiated condition, andthe amount released varies depending thereon. In the present invention,an attention is directed to the contents of oxidative glutathione andreductive glutathione in macrophages. Macrophages are monitored by theratio of oxidative macrophages and reductive macrophages, and theimmunological state is identified. The balance of these macrophages ismodulated with the immunomodulator and the like of the present inventionto improve the in vivo immunological state and to treat or preventvarious diseases usefully.

In the reductive macrophage, the content of reductive glutathione isrelatively higher than that in oxidative macrophage. In the oxidativemacrophage, the content of reductive glutathione is relatively lowerthan that in reductive macrophage. Further, the reductive macrophage andthe oxidative macrophage are different in activation of atranscriptional factors due to the difference in the reductive GSHcontent. Consequently, there occurs a difference in the gene expressionof cytokines or inflammatory mediators, so that the type and the amountof the resulting inflammatory cytokines or inflammatory mediators arechanged and the quality of inflammation is changed.

With the oxidative macrophage, inflammatory cytokines and mediators suchas IL-6, IL-1, IL-8, IL-10, TNF, hydrogen peroxide, superoxide and PGE2are produced. With the reductive macrophage, nitrogen monoxide (NO),IL-12 and LTB4 are produced. Further, the oxidative macrophage and thereductive macrophage are inter-converted through stimulation or thelike. The reductive macrophage can be converted to the oxidativemacrophage through artificial stimulation using LPS or PMA inducinginflammatory or ischemic shock and cytokines such as IL-4 and TGFβ. Onthe contrary, the oxidative macrophage can be converted to the reductivemacrophage with the addition of IFNγ, IL-2, lentinan (LNT) which is anantitumor polysaccharide, or lipoic acid with an antioxidant nature.This can be applied to therapy of immunological diseases.

The amounts of the oxidative macrophage and the reductive macrophagevary depending on the pathological state of the said diseases. Theamount of the oxidative macrophage contained in the body fluid or thecell sample collected from patients suffering from allergic diseases oradvanced cancers is relatively larger than that in the healthy person.This can be used in the examination for diagnosis of immunologicaldiseases and tumor cachexia and the subsequent therapy thereof.

Further, it has been clarified through image analysis using an adherentcell analyzing system (ACAS) or biochemical determination using anenzyme recycling method that a content of reductive glutathione inmacrophages harvested from model animals suffering from gastrointestinalinflammatory diseases (hepatitis, Crohn disease and ulcerative colitis)is relatively lower than that in normal animals. This indicates thatmacrophages are inclined to be oxidative in intestinal inflammatorybowel diseases or gastrointestinal inflammatory diseases, and thatoxidative macrophages participate in a mechanism of preventing theprogression of diseases. When oxidative macrophages participate in thedisease progression, these have to be converted to reductive ones. Whenoxidative macrophages participate as a defense mechanism of preventingdisease progression, the oxidative state of macrophages has to bemaintained through administration of a drug. The inventors haveassiduously conducted studies as to which of these two possibilities isthe essence of diseases. Consequently, they have found for the firsttime in the world that the oxidative macrophages have the defensemechanism of preventing the progression of chronic inflammatory diseasesand autoimmune organ specific inflammatory diseases mainly in thedigestive tract, and that it is useful to reduce the content ofreductive glutathione in the macrophages as an approach of improving andtreating the gastrointestinal inflammatory diseases.

In accordance with the present invention, the low molecular weightcompound which has an activity of changing the content of reductiveglutathione in the macrophage cell after measuring the same by theabove-mentioned method and which maintains the activity even through theoral intake is formulated into a drug in a usual manner, and this drugcan be taken in the patient every day or at fixed intervals uponmonitoring the condition of the disease. At the chronic stage, themarked effect is brought forth by the long-term administration.

With respect to the definition of the oxidative macrophage and thereductive macrophage in the present invention, the reductive glutathione(GSH) content in the cell is determined through the reaction withmonochlorobimane which is a chemical reagent specific to GSH. Themacrophage of which the GSH content is increased in comparison with theresident macrophage is defined as the reductive macrophage, while themacrophage of which the GSH content is decreased is defined as theoxidative macrophage. Preferably, the macrophage of which the GSHcontent is more than 2 nmoles/5×10⁵ macrophages by being brought intocontact with the low-molecular weight substance capable of oral intakefor from 2 to 24 hours is defined as the reductive macrophage, and themacrophage of which the GSH content is less than 0.1 nmoles/5×10⁵macrophages is defined as the oxidative macrophage. Alternatively, themacrophage of which the GSH content is at least twice that of theresident macrophage is defined as the reductive macrophage, while themacrophage of which the GSH content is at most ⅕ that of the residentmacrophage is defined as the oxidative macrophage.

As the substance having the activity of changing the content ofreductive glutathione in macrophages, any substance will do ifmacrophages (or monocytes) are incubated at concentrations of 5×10⁵cells/200 μl/well using a 96well microplate, from 0.01 μM to 5 mM of asubstance to be tested are added thereto and incubated at 37° C. in a 5%CO₂ incubator and the reductive GSH content is increased or decreasedrelative to the control group after from 2 to 24 hours. A substance thatcan increase the GSH content to 2 nmoles/5×10⁵ macrophages or more ordecrease the same to 0.1 nmoles/5×10⁵ macrophages or less is preferable.Examples thereof include antioxidants, for example, a precursor of GSHwhich is metabolized into GSH in cells, such as N-acetylcysteine (NAC),-glutamylcysteine diethyl ester, glutathione derivatives such asglutathione monoester and glutathione diester, lipoic acid andderivatives thereof, ortene, and flavonoid and derivatives thereof. Theyare substances having an activity of changing the content of glutathionein cells by the incubation with macrophages in vitro for a few hours.These agents can be used either singly or in combination. The effectthereof can be measured by collecting monocytes from an body fluid oflocal inflammatory sites or a peripheral blood after the intake or theadministration and determining the change in the content of reductiveglutathione in cells relative to that before the treatment by theabove-mentioned method. The usefulness as the immunomodulator is clearlyevaluated by this procedure, and the agents are effective for thetreatment of the patients. When a cysteine derivative is used as animmunosuppressant, it is included by the above-stated immunomodulatorand as a matter of course, it can be used in the same way as statedabove.

The diseases to which the present invention is applied include cachecticconditions of patients suffering from cancers; autoimmune diseases suchas diabetes, chronic rheumatoid arthritis, SLE and pulmonary fibrosis;inflammatory diseases such as hepatitis, hepatic cirrhosis andinflammatory bowel diseases, centering on gastrointestinal inflammatorydiseases; and allergic diseases such as hypersensitive interstitialpneumonia, asthma, atopic cutaneous inflammatory diseases, sarcoidosis,etc.

Especially, as diseases to which the immunosuppressant according to thepresent invention is applicable, it is expected that it is effectiveagainst autoimmune inflammatory diseases. Above all, it is desirable toapply the immunosuppressant to chronic inflammatory diseases caused indigestive organs, including a group of diseases called inflammatorygastrointestinal diseases such as hepatitis, hepatic cirrhosis andinflammatory bowel diseases such as ulcerative colitis and Crohndisease.

The agents can widely be applied to diseases associated with theabnormal Th1 /Th2 balance or the functional deficiency of macrophages,for example, cachexia of patents suffering from cancers, diabetes,chronic rheumatoid arthritis, autoimmunological diseases such as SLE,chronic inflammatory diseases such as hepatitis, hepatic cirrhosis,inflammatory bowel diseases, and allergic diseases such as asthma,cutaneous atopy and sarcoidosis. The agents are also effective forchemoprevention of cancers as the immunomodulator. This makes it clearthat during the period in which one normal cell undergoes transformationand carcinogenesis in the human body and then reaches to 10⁹ cells wherethe presence of cancer tissues is clinically detected, the canceroustissue is profitably present in the reductive condition. That is, it isscientifically verified that active oxygen or the like which is producedby inflammatory responses in vivo contributes to malignant progression.

The immunomodulator used in the present invention can be administeredsingly in the actual medical care. The immunomodulators capable of oralintake which are included in present invention can also be used incombination. Further, immunomodulator of the present invention can bemixed with, or used in combination with, the other immunomodulator incapable of oral intake but charging the content of reductive glutathionein macrophages with the different function, for example, exogenous andendogenous substances such as β(1-3) glucan typified by lentinan andcytokines typified by interleukin 2(IL-2). Especially when it isrequired to increase cellular immunity, IL-2 or γ-interferon (γIFN) isused in combination whereby interleukin 12 (IL-12) is produced in vivoin a large amount from the reductive macrophage to more increase theeffect the present invention. On the other hand, when the therapeuticeffect is intended by decreasing cellular immunity, production of IL-12is decreased with the combined use of interleukin 4 (IL-4) or TGFβ toincrease the effect. It has been found in the present invention thatthese cytokines change themselves the content of reductive glutathionein macrophages, increasing the usefulness and the scope of the presentinvention.

When substances which inhibit the production or the function of IL-12,other than antibodies, are used in combination as in vitro substances, asynergistic effect is further expected.

It is also included in the present invention that either of themacrophages which are different in the content of reductive glutathionein cells, namely, the macrophage (oxidative macrophage) having the lowreductive GSH content and macrophage (reductive macrophage) having thehigh reductive GSH content is selectively removed. The substance used inthis case may be a low-molecular weight compound or a high-molecular hatcompound. Among others, antibodies and derivatives thereof areeffective.

As already stated, the correspondence of a variety of functions ofmacrophages/monocytes to their subsets has been to date totally unknown.Accordingly, although macrophages/monocytes play quite an important rolein the triggering and the progression of inflammatory diseases, allergicdiseases and immunological diseases, the functional classification onthe basis of the presence of distinct macrophage/monocyte sub-sets hasnot been applied at all to the therapy, improvement and prevention ofhuman diseases, and this application has not been even imagined. Beforethe completion the present invention, the reductive GSH content of themacrophage was measured, and it was discovered for the first that thereis a great difference in an effect of macrophages having different GSHcontents on the immunological functions. Further, the contents ofoxidative glutathione and reductive glutathione in macrophages whichplay an important role in the inflammatory reaction were measured toclassify heterogeneous macrophages into the two types, namely, oxidativemacrophages and reductive macrophages. Then, it was found that theoxidative macrophages induce local chronic inflammatory diseases orallergic reaction accompanied by immunological diseases, that theTh1/Th2 balance controlling the balance of humoral immunity and cellularimmunity is regulated by the redox state of macrophages, end that theredox state of the macrophages plays an important role in theprogression of immunological diseases. In order to artificially controlthe presence ratio of these two macrophages, the above-mentionedlow-molecular weight substance capable of oral intake is used as a drug,and the selective removal of either of these macrophages is also quiteuseful. This is also understandable from the fact that variousmonoclonal antibodies to lymphocytes are on the market as animmunosuppressor. It is easily conceivable to those skilled in the artthat antibodies to either of these macrophages or to markers expressedin larger amounts in either of these macrophages can be used.

Further, substances having toxicity to cells or derivatives thereof canbe used. However, since there is a great difference in intracellularenzymatic activities between reductive-macrophages and oxidativemacrophages, substances which can be converted to those having aselective cytotoxicity within either of reductive macrophages oroxidative macrophages are most appropriate prodrugs in the presentinvention. For example, the use of a pyrimidine nucleotide phosphorylaseenzymatic activity or a glutathione-S-transferase enzymatic activitywhich is increased in the oxidative macrophages is mentioned. There is aproduct in which an alkylating agent having a cytotoxicity is conjugatedwith glutathione.

That the immunomodulator of the present invention can be applied to awide variety of immunological diseases is clearly seen from the factthat it controls the secretion of an inflammatory mediator frommacrophages at the very beginning stage of the production. For example,non-steroidal acidic anti-inflammatory drug (aspirin or the like) issaid to exhibit the pharmaceutical effect by controlling production orsecretion of prostaglandin. Meanwhile, an antioxidant such as vitamin Eexhibits the pharmaceutical effect by controlling production of activeoxygen. Thus, the function is only to control one of various propertiesof macrophages which are inflammatory cells. For this reason, its effectis not workable, and almost no effect is exhibited to chronicinflammatory diseases in particular. On the other hand, theimmunomodulator of the present invention controls the redox condition ofmacrophages, and can suppress the production of a large number ofharmful inflammatory mediators all at once. In this context, theconventional concept to date of anti-inflammatory drugs is said to befundamentally changed.

As stated above, the useful pharmaceutical effect of the immunomodulatorof the present invention in the actual medical care is self-evident fromits profitable immunological activity. It is useful for both the acuteand chronic stages of diseases. Especially, it can widely be applied todiseases associated with the abnormal Th1/Th2 balance or the functionaldeficiency or the abnormality of macrophages, for example, cachexia ofpatients suffering from cancers; autoimmunological diseases such asdiabetes, chronic rheumatoid arthritis, SLE and pulmonary fibrosis;chronic inflammatory diseases such as hepatitis, hepatic cirrhosis andgastrointestinal inflammatory diseases centering on inflammatory boweldiseases (ulcerative colitis and Crohn disease); and allergic diseasessuch as hypersensitive interstitial pneumonia, asthma, cutaneous atopyand sarcoidosis . It is also effective for chemoprevention of cancers.With respect to the cachectic condition of patients suffering fromcancers, an effect to increase the survival rate is expected, and theimmunomodulator is considered to be also useful especially in theimprovement of quality of life (QOL) of the patient.

Especially, with respect to the immunomodulator of the invention, theuse of the cystine derivatives as an immunosuppressant is described indetail below.

The substances having the activity of reducing the content of reductiveglutathione are as mentioned above. The cystine derivatives representedby the structural formula (1) and having the activity of reducing thecontent of reductive glutathione in the macrophages are all included inthe cystine derivatives used in the invention. Examples thereof includeN,N′-diacetylcystine-[(NAC)₂], N,N′-dipropylcystine [(NPC)₂],N,N′-diacetylcystinedimethyl ester [(NAC-OMe)₂],N,N′-diacetylcystinediisopropyl ester [(NAC-OiPr)₂] andN,N′-di-L-alanylcystinedimethyl ester [AlaC-OMe)₂.

With respect to R¹ and R² in the structure of the compounds, a widevariety of substituents can be used so long as reductive glutathione inthe macrophages is oxidized into oxidative macrophages through thedisulfide linkage. For example, R¹ and R², independently from eachother, represent an alkyl group having from 1 to 12 carbon atoms(inclusive of all specific values and subranges therebetween, 1, 2, 3,4, 5, 6, 7, 8, 9, 10 and 11 carbon atoms) or a substituted alkyl groupsuch as the nitroxybutyl group, and R³ and R⁴, independently from eachother, represent an acyl group having from 1 to 12 carbon atoms(inclusive of all specific values and subranges therebetween, 1, 2, 3,4, 5, 6, 7, 8, 9, 10 and 11 carbon atoms) or a peptidyl group. Thepeptidyl group means an amino acid residue or peptide residue consistingof plural amino acids, which is bound through its carboxyl group. Thenumber of amino acid may vary widely, for example, from 1 to 10 aminoacids. Preferably, the acyl groups are derived from hydrocarboncarboxylic acids.

These agents can be used either singly or in combination. The effectthereof can be estimated by harvesting monocytes from inflammatory partsor peripheral blood after intake or administration, examining the changein the content of reductive glutathione in cells relative to that beforetreatment using the method and measuring the change in the immunologicalactivity in vivo. This clearly proves the usefulness as theimmunosuppressant in particular, and these agents are effective againstdiseases.

The administration form is not particularly limited, and it includesadministration by injection and oral administration.

However, the oral administration is advantageous. The dose of thesubstance having an activity of changing the content of reductiveglutathione as an active ingredient is selected depending on theconditions of patients or the like to which the substance isadministered or the use purpose. In the case of patients suffering fromserious diseases, for example, a advanced gastric cancer, the dose isbetween 1 and 5,000 mg (oral drug), preferably between 10 and 500mg/day. It is not particularly difficult to produce preparations, andpreparations can be produced in the form of an oral agent, an injection,a percutaneous agent and the like as required in a usual manner.

It has been described above that the immunomodulator of the presentinvention is quite useful and quite new as a drug im a narrow sense.Since the immunomodulator of the present invention contains a substancecapable of oral intake as a main ingredient, its use is not limited todrugs in the actual medical care. That is, the immunomodulator of thepresent invention can also be provided in the form of a food (includingall that are put into the mouth, such as a chewing gum, a tooth pastethe like), as a food for medical care, a health food or a specialsanitary food containing a substance having an activity of changing thecontent of reductive glutathione in human macrophages (includingmonocytes, Kupffer cells and dendritic cells) either singly or as amixture, as well as in the form of a nutrient an infusion. These arealso included in the present invention. It can also be contained in aliquid component or take the form of a solid food.

The food, the nutrient and the infusion can be applied to the samediseases as those to which the drugs are applied.

The immunomodulator of the present invention can be provided in the formof the food, the nutrient and the infusion having an immunomodulatoryfunction for improvement of the cachectic condition of patientssuffering from cancers, diabetes, inflammatory bowel diseases, chronicrheumatoid arthritis, hepatitis, hepatic cirrhosis, gastrointestinalinflammatory diseases centering on inflammatory bowel diseases(ulcerative colitis and Crohn disease), autoimmune inflammatorydiseases, and for chemoprevention of cancer. The dose of the activeingredient may be determined according to what has been described in theabove-mentioned drugs. It can be applied not only to patients sufferingfrom attacked or chronic diseases but also to high-risk personssuffering from adult diseases or the like.

Having generally described this invention, a further understanding canbe obtained by reference to certain specific examples which are providedherein for purposes of illustration only and are not intended to belimiting unless otherwise specified.

EXAMPLES Example 1

Test for functions of oxidative macrophages and reductive macrophages

Oxidative macrophages were induced by administering 20 μg of LPS(lipopolysaccharide) to an abdominal cavity of a mouse, and reductivemacrophages were induced by administering 100 μg of lentinan to anabdominal cavity of a mouse three times every two days. These wereclarified by adhering peritoneal exudate cells to the plastic surface,then reacting the same with 10 μM of monochlorobimane at 37° C. for 30minutes and conducting analysis with Adherent Cell Analyzing System(ACAS). The increase in the amounts of oxidative macrophages can easilybe measured visually from the fact that almost no reaction product isobserved, that is, gray or blue image is obtained, and the increase inthe amounts of reductive macrophages from the fact that the red oryellow image is obtained, respectively.

Accordingly, NO, IL-6 and PGE2 produced by inducing the peritonealexudate cells into oxidative end reductive cells were measured.

(1) Materials

Cells: The peritoneal exudate cells obtained by the above-mentionedstimulation, namely, the macrophages were added to a 96-well microplatein an amount of 1×10⁵ cells/200 μl each.

Medium: Phenol red-free RPM 11640: 200 μl/well

LPS: Lipopolysaccharide (made by Sigma Co.) (origin: E. coli) 100 ng/ml

IFNγ: 100 units/ml

(2) Incubation

Incubated in a 5% CO₂ incubator at 37° C. for 48 hours.

(3) Measuring method

After the completion of the above-mentioned incubation, the culturesupernatant was recovered. The amount of IL-6 was measured by theproliferation assay using an IL-6-dependent cell strain, MH60, theamount of PGE2 was measured using an ELISA-kit, and the amount of NO wasmeasured using a Griess-Romijn reagent. These measurements wereconducted by a method which those skilled in the art usually employ.

(4) Results:

The results are shown in FIG. 3. As is clear from FIG. 3, there aredifferences in the concentration and the type among inflammatorycytokine IL-6, inflammatory mediator PGE2 and NO produced betweenoxidative macrophages and reductive macrophages. That is, with theoxidative macrophages, the production of IL-6, a Th2 cytokine and theproduction of PGE2, which is immunosuppressive to suppress the Th1induction are increased, and the production of NO is decreased. On thecontrary, with the reductive macrophages, the production of NO isincreased, and the production of PGE2 and the production of IL-6 aresuppressed. Thus, there is a functional difference seen between bothmacrophages.

Example 2

Test using animal disease models which are immunologically deficient byknocking out a gene

In order to clarify a mechanism underlying the conversion of an acute toa chronic phase and progression of inflammatory diseases, it isimportant to analyze molecularly why there is a difference in theproduction of an inflammatory mediator or a cytokine between oxidativeMΦ and reductive MΦ. Generally, extracellular stimulation (ligand or thelike) of the all is signaled into cells through a receptor present onthe cell surface. Various kinases are activated with signals from thereceptor, and transcriptional factors are also activated in cytoplasm.The activated transcriptional factors are translocated into the nucleus,and bound to target genes to conduct gene expression. According to therecent studies, it is being clarified that the intracellular redoxsystem regulates activation of transcriptional factors, translocationthereof into the nucleus and binding with genes (Annual Rev. Immunology,vol. 8, pp. 453-475, 1990, Embo J., 10, 2247-2251, 1991). It iscurrently unknown how the intracellular redox system participates in thegene expression system after the receptor triggering in MΦ. As a methodof clarifying the same, MΦ was harvested from a knock out mousedeficient in a molecule participating in a signal transduction systemfrom a receptor, and the function of the redox state was analyzed.Specifically, a common γ chain (γc) which is commonly used as a receptorconstituting molecule of IL-2, IL-4, IL-7, IL-9 and IL-5 and Jak3 whichis a molecule present downstream thereof and transducing a signal fromγc were used as target molecules.

Cytokine and stimulator:

As mouse IFNγ, a recombinant supplied by Genzyme was used. As human IL-2and human IL-6, recombinants supplied by Ajinomoto Co., Inc. were used.As human IL-12, a recombinant supplied by Pharmingen was used.

As LPS, a substance derived from E. coli 055:B5 supplied by Difco wasused. As lentinan, a preparation produced by Ajinomoto Co., Inc. wasused.

Mice used:

γc Knock out mice were obtained from Professor Sugamura, TohokuUniversity Medical School. Jak3 knock out mice were obtained fromProfessor Saito, Chiba University Medical School.

As wild mice used for mating and as a control, C57BL/6 obtained fromCharles River Japan (CRJ) was used.

Harvest of peritoneal MΦ:

Peritoneal cells were harvested by injecting 5 ml of a phenol red-freeDMEM medium (supplied by Nikken Seibutsusha) ice-cooled into aperitoneal cavity of a mouse which had been put to sacrificial deathwith ether using an injection cylinder fitted with a 22-gauge needle,squeezing the same and pulling out the medium.

Determination of the amount of IL-6:

A stimulator was added to 1×10⁶ MΦ, and the incubation was conducted at37° C. for 2 days in a CO₂ incubator. After centrifugation, the culturesupernatant was collected.

The amount of IL-6 was determined using IL-6 dependent mouse hybridomaMH60 cells (J. Eur. Immunol., vol. 18, p. 951, 1988). One hundredmicroliters of the culture supernatant were added to 100 μl of the MH60cell suspension adjusted to 1×10⁵ cells/ml in a 10% FCS-containing RPMImedium, and the mixed solution was incubated at 37° C. for 2 days in aCO₂ incubator. Subsequently, 10 μl of MTT (supplied by Sigma Co.)solution adjusted to a concentration of 5 mg/ml in the same medium wereadded thereto, and the reaction was conducted at 37° C. for 5 hours.After the completion of the reaction, the centrifugation was conducted.The supernatant (160 μl) was removed, and 100 μl of a mixture ofhydrochloric acid and propanol were added to the residue. The suspensionwas conducted using a pipetman to dissolve the cells. Immediately afterthe dissolution, an absorbance of 570 nm was measured with animmunometer (supplied by Bio-Rad).

Measurement of a concentration of NO₂:

A stimulator was added to 1×10⁵ MΦ, and the incubation was conducted at37° C. for 2 days in a CO₂ incubator. After the completion of thecentrifugation, the culture supernatant was collected.

One hundred microliters of a Griess-Romijn reagent (supplied by WacoPure Chemical Industries, Ltd.) adjusted to a concentration of 50 mg/mlin distilled water were added to 100 μl of the culture supernatant, andthe reaction was conducted at room temperature for 15 minutes. After thecompletion of reaction, an absorbance of 540 nm was measured. NaNO₂ wasused as a standard.

Determination of GSH in cells with ACAS:

Three-hundred microliters of a cell suspension adjusted concentration of3×10⁵ cells/ml in an RPMI 1640 medium (phenol red-free) were chargedinto a chambered coverglass (#136439, supplied by Nunc), and incubatedat 37° C. for 2 hours using a CO₂ incubator. The culture solution waswashed with the medium, and 300 μl of monochlorobimane (supplied byMolecular Plobe) adjusted to 10 μM in the same medium were addedthereto. The mixture was charged into a CO₂ incubator of 37° C., and thereaction was conducted for 30 minutes. The fluorescent intensity wasmeasured with ACAS. In ACAS, a UV laser was used.

Determination of an amount of IL-12:

The amount of IL-12 was determined through bioassay using cells of humanT cell strain 2D6 (J. Leukocyte Biology, vol. 61, p. 346, 1997).

2D6 cells which had been incubated in an RPMI 1640 medium containing 500pg/ml of recombinant human IL-12, 50 μM of 2-mercaptoethanol and 10% FCS(fetal calf serum) were moved to a tube, and centrifugally washed threetimes with the above-mentioned medium without IL-12 and cell density wasadjusted to 1×10⁵/ml. The cell suspension was added in an amount of 100μl each to a 96-well flat bottom plate containing a sample seriallydiluted in advance with an RPMI 1640 medium containing 50 μM of2-mercaptoethanol and 10% FCS in an amount of 100 μl each. Subsequently,the mixture was charged into a 5% O₂ incubator of 37° C., and incubatedfor 48 hours. For final 6 hours, ³H-TdR was pulsed (a substance adjustedto 370 kBq/ml in an RPMI 1640 medium containing 50 μM of2-mercaptoethanol and 10% FCS was added in an amount of 50 μl each). Thecells a harvested, and the radioactivity was measured using a P counter(Matrix 96, supplied by Packard).

Measurement of the GSH content in MΦ produced from knock out mice:

Peritoneal cells were produced from knock out mice, and the GSH contentin cells was analyzed by ACAS using an MCB reagent. The content ofreductive glutathione was clearly decreased in any mice compared withcontrol mice (C57BL/6).

Function of MΦ produced from knock out mice:

Peritoneal cells were produced from wild mice (C57BL/6) knock out mice,and stimulated with LPS, IL-2, IFNγ and a combination thereof. The NOproduction, the IL-6 production the IL-12 production were measured.Almost no NO production was observed in any mice derived MΦ in theabsence of stimulation. In the stimulation with the combination of LPSIFNγ, almost no additive effect was observed in the γc knock out mice,and the NO production was decreased to less than half that in controlmice. The same results as in γc were provided in Jak3 knock out mice.Further, the IL-6 production was analyzed. In the LPS stimulation, anincrease in the IL-6 production was observed in γc knock out mice (962pg/ml relative to 81 pg/ml of a control). In the IFNγ stimulation, anincrease in the IL-6 production was observed in γc knock out mice. Theresults were the same with the suppression pattern of the NO production.Still further, the IL-12 production with the LPS stimulation and theIFNγ stimulation was examined. No production was observed at all in anymice derived MΦ. This proves that in the sick animals of the gene knockout mice used herein, the amount of the oxidative macrophages isincreased to increase the humoral immunity or the allergic reactionmainly caused by Th2 and to decrease the cellular immunity supported byTh1. In the animal disease models, it is clearly shown that thediagnosis of immunological diseases required for the immunomodulator ofthe present invention is original and significant.

Example 3

Determination of the amount of reductive glutathione in MΦ of advancedtumor-bearing mice

Method:

Oxidative and reductive macrophages collected from peritoneal cavitiesof advanced tumor-bearing cachectic mice (COLON 26) and normal mice weredetermined. The COLON 26 transplantable tumor well known to induce acancer cachexia was implanted subcutaneously in the back portion of CDF1mice at a density of 5×10⁵ cells/mouse. On day 21 after the tumorimplantation, the cachectic condition was provided. Five milliliters ofa physiological saline solution were intraperitoneally injected into themice which became resistant to a therapeutic treatment. Peritonealmacrophages were collected, and suspended in a phenol red-free RPMI 1640medium containing 10% fetal calf serum to a density of 3×10⁵ cells/ml.One hundred microliters of the suspension were charged in a Lab-TekChamber Slide (#136439, supplied by Nunc), and incubated in 5% CO₂ at37° C. for 3 hours. After the nonadherent cells were removed, 200 μl ofthe above-mentioned medium free from serum were added thereto, andmonochlorobimane (MCB) was added thereto in an amount of 10 μM. Thereaction was conducted for 30 minutes, and the image analysis wasconducted on the basis of the UV absorption using an ACAS device(supplied by Meridien).

Results:

The content of reductive glutathione was determined by ACAS. As aresult, in the advanced tumor-bearing mice, the amount of the macrophageof which the reductive glutathione content was decreased, namely, theoxidative macrophage was relatively increased in comparison with that inthe normal mice. Since the amount of the oxidative macrophage wasincreased, the amount of IL-6 in the above-mentioned macrophage culturesupernatant was markedly increased (600 pg/ml relative to 120 pg/ml incontrol mice). Further, the amount of PGE 2 was 32 ng/ml relative to 7.6ng/ml in control mice, and it was increased tp 5 times or more. It wasfound that the immunosuppressive state or the cachectic state at theadvanced tumor-bearing stage is based on the excessive production ofthese mediators. In addition, the increase in an amount of active oxygenproduced was also observed. It shows that the redox state of macrophagesis measured upon determination of the glutathione content withoutmeasuring a large number of parameters whereby the examination fordiagnosis of the pathological state and the immunological function ofpatients suffering from cancers can be conducted easily and exactly.Accordingly, the above-mentioned classification of macrophages enablesthe examination for diagnosis of diseases and the immunological functionof patients suffering from cancers.

Example 4

Induction of reductive macrophages by oral administration of glutathioneethyl ester to advanced tumor-bearing mice

The COLON 26 transplantable tumor was implanted subcutaneously in theback portion of CDF1 mice at a density of 5×10⁵ cells/mouse. On day 21after the tumor implantation, the mice were proved to be in thecachectic condition. Glutathione ethyl ester was orally administered tothe mice every day in a dosage of 1 mg/0.5 ml/h. This oraladministration was continued for 10 days. The peritoneal cells werecollected from the mice in the same manner as in Example 3. Peritonealmacrophages were collected, and suspended in a phenol red-free RPMI 1640medium containing 10% fetal calf serum to a density 3×10⁵ cells/ml. Thesuspension was charged into a Lab-Tek Chamber Slide (#136439, suppliedby Nunc) in an amount of 100 μl, and the incubation was conducted in 5%CO₂ at 37° C. for 3 hours. After the nonadherent cells were removed, 200μl of above-mentioned medium free from serum were added thereto, and 10μM of monochlorobimane were then added thereto. The reaction wasconducted for 30 minutes, and the image analysis was conducted based onthe UV absorption using an ACAS device (supplied by Meridien).

Results:

The content of reductive glutathione was determined by ACAS method.Consequently, in the advanced tumor-bearing model mice to whichglutathione ethyl ester had been administered, the amount of themacrophage of which the reductive glutathione content was decreased,namely, the oxidative macrophage was relatively decreased in comparisonwith that in control mice to which the physiological saline solution hadbeen administered. Since the amount of the reductive macrophage wasincreased, the amount of IL-6 in the above-mentioned macrophage culturesupernatant was decreased (642 pg/ml relative to 5,200 pg/ml in controlmice). Further, the amount of PGE2 was also much decreased to 6.5 ng/mlrelative to 32 ng/ml in control mice. It was thus clarified that theimmunosuppressive state or the cachectic state at the advancedtumor-bearing stage can be improved by the oral administration ofglutathione ethyl ester. Accordingly, the average number of survivaldays of mice in the treated group increased from 42 (in control mice) to148.

Example 5

Examination of macrophages collected from the patient suffering fromsarcoidosis and conversion of oxidative macrophages to reductivemacrophages

The amounts of oxidative and reductive monocytes macrophages containedin monocytes preparation separated and collected in a usual manner fromthe peripheral blood and the thoracic cavity of the patient sufferingfrom sarcoidosis were examined by biochemically measuring the contentsof reductive glutathione (GSH) end oxidative glutathione (GSSG) by theenzyme recycling method. The peripheral blood of the healthy person wasused as a control.

Materials:

The peripheral blood of the healthy person and the peripheral blood ofthe patient suffering from sarcoidosis were collected with heparin. Or150 ml of a physiological saline solution was injected into the bronchiaof the patient using a bronchofiber, and 75 ml of bronchoalveolarlavaged fluid were recovered. Monocytes obtained by separating andpurifying both of them using Ficoll-Hypaque (LYMPHOPREP) were suspendedin an RPMI 1640 medium containing 10% fetal calf serum, and washed threetimes to obtain macrophage/monocyte preparation adherent to a glasspetri dish for 30 minutes. Subsequently, a group incubated for 3 hourswith the addition of 5 mM N-acetylcysteine (NAC) and a group of a mediumcomponent alone were prepared. A rubber policeman was used to recoveradherent cells from the petri dish. With respect to 5×10⁵ macrophages,the following examination was conducted.

Method:

The contents of reductive glutathione and oxidative glutathione weremeasured by the above-mentioned enzyme recycling method.

Production of samples:

One hundred microliters of Triton X-100 prepared with a 0.1M phosphatebuffer (pH 7.5) containing 5 mM EDTA ice-cooled were added to cellpellets which had been washed with PBS, and the mixture was allowed tostand at room temperature for 5 minutes to dissolve the cells. Fifteenmicroliters of 0.1M HCl were added thereto, and 15 μl of a 50%sulfosalicylic acid (SSA) solution were further added thereto. Themixture was centrifuged at 12,000 rpm for 5 minutes, and the supernatantwas collected [*] to form a measuring sample having a total glutathioneconcentration (GSH+GSSG).

Measuring method:

A 10 mM phosphate buffer (590 μl, pH 7.5) containing 0.5 mM EDTA, 100 μlof glutathione reductase (supplied by Boehlinger Mannhein) adjusted to aconcentration of 6 u/ml in the same buffer, 50 μl of 4 mM NADPH(supplied by Sigma Co.) prepared with 5% NaHCO₃ and 10 μl of the samplewere mixed. The mixture was incubated at 37° C. for 5 minutes. Fiftymicroliters of a 10 mM 5,5′-dithio-bis(2-nitrobenzoic acid) (DTNB,supplied by Sigma Co.) prepared with a 0.1M phosphate buffer (pH 7.5)containing 5 mM EDTA were added thereto. The change in the absorbance of412 nm at 37° C. over the course of time was measured using aspectrophotometer. As a standard sample, GSH (supplied Sigma Co.)prepared in the same manner as the above-mentioned sample was used.Separately, the content of oxidative glutathione (GSSG) alone wasmeasured, —2 μl of 2-vinylpyridine (supplied by Tokyo Kaseisha) wereadded thereto after the above-mentioned procedure [*] and were mixed atroom temperature for 1 minute, and after the pH was adjusted to 7.5,reaction mixture was allowed to stand at room temperature 60 minutes toform a measuring sample, and the measurement conducted in theabove-mentioned manner. —and the content of reductive glutathione (GSH)was obtained by the subtraction from the total glutathione content.

Results:

With respect to the contents of reductive glutathione and oxidativeglutathione in the peripheral blood of the patient, the GSSG content was5.29 μM, and the GSH content was 20.45 μM. Thus, the ratio of reductiveGSH was approximately 80%, and still higher (the ratio of reductive GSHwas 90% or more in the healthy person). In the macrophages within thethoracic cavity, the content of reductive GSH was 1.45 μM, and thecontent of oxidative GSSG was 15.85 μM. Thus, the ratio of oxidativeGSSG was approximately 86%, and the presence ratio thereof wascompletely inverted. In the NAC addition group, content of reductive GSHwas 20.45 μM, and the content of oxidative GSSG was 4.32 μM. Thus, thecontent of oxidative GSSG was largely decreased, and the ratio ofreductive GSH exceeded 80%. In this manner, the peripheral blood levelwas recovered. It shows that in this disease, the oxidative macrophagesplay a great role in the progression of the disease and this progressioncan be improved through NAC administration. Thus, the usefulness of thepresent invention is clarified not only in animal disease models butalso in patients suffering from rheumatoid arthritis or diabetes andpeople with a high risk of these diseases.

Example 6

Induction of reductive macrophages by oral administration of NAC andGSH-OEt

Macrophages (MΦ) were prepared from knock out mice deficient in amolecule participating in a signal transduction system from a receptor,and the function of the redox system was analyzed. Specifically, acommon γ chain (γc) which is commonly used as a receptor constitutingmolecule of IL-2, IL4, IL-7, IL-9 and IL-15 and JaK3, a molecule presentdownstream thereof and transducing a signal from γc were gene targetedmolecules. Methods applied in Example 2 was repeated. JaK3 knock outmice were divided into three groups. A control group was a group ofusual city water free-intake. An NAC group was a group of free intake ofcity water containing 1 mg/ml of NAC. A GSH-OEt group was a group offree intake of city water containing 1 mg/ml of GSH-OEt. Breeding wascontinued under SPF condition for 24 days, and peritoneal exudate cells,namely, macrophages were likewise obtained.

Cytokines and stimulator:

A recombinant supplied by Genzyme was used as mouse IFN. Recombinantssupplied by Ajinomoto Co., Inc. were used as human IL-2 and human IL-6.A recombinant supplied by Pharmigen was used as human IL-12.

A product derived from E. coli. 055; B5 as supplied by Difco was used asLPS. A preparation supplied by Ajinomoto Co. Inc. was used as lentinan.

Determination of an amount of IL-6:

Measurement of an NO₂ concentration:

Determination of a GSH content in cells by ACAS:

Determination of an amount of IL-12:

These were all conducted in the same manner as in Example 2.

Measurement of a GSH content in MΦ prepared from knock out mice:

Peritoneal cells of knock out mice which had undergone the respectivetreatments were harvested, and the GSH content in the cells was analyzedusing ACAS with MCB. In any of the mice in the groups of free intake ofcity waters containing NAC and GSH-OEt, the content of reductiveglutathione in MΦ was markedly increased in comparison with that incontrol mice (city or free intake group). The image of reductive MΦderived through intraperitoneal administration of NAC in normal mice wasshown.

Function of MΦ harvested from knock out mice:

Peritoneal cells were harvested from three groups of knock out mice, andstimulated with LPS, IL-2, IFNγ and a combination thereof The NOproduction, the IL-6 production and the IL-12 production were measured.With respect to the NO production, almost no NO production was observedin any mice derived MΦ in the absence of stimulation. Then, the IL-6production was analyzed. In LPS stimulation, the amount was detected as962 pg/ml in the knock out mice derived MΦ culture, 122 pg/ml in the NACgroup, and 82 pg/ml in the GSH-OEt group. In view of the function, itwas identified that the conversion to reductive macrophages waspossible. In consideration of the fact that IL-6 is a main cytokine ofinducing Th2, it is clearly shown that the biological Th1/Th2 balancecan be controlled by the oral intake of these substances. This resultwas inversely related with the suppression of the NO production and therecovery pattern with medications. Next, the IL-12 production bystimulation of LPS or IFNγ was examined. No production was observed atall in the control group. This shows that in the animals disease models,the JaK3 gene knock out mice used here, the amount of the oxidativemacrophage is increased, humoral immunity or an allergic reaction mainlycaused by Th2 is increased, and cellular immunity caused by Th1 isdecreased. On the other hand, it was identified that in the NAC andGSH-OEt administration groups, the amounts of IL-12 are 420 pg/ml and610 pg/ml respectively. This proves that in the animal disease models,the immunomodulator of the present invention is also useful in theimprovement of the immunological diseases, and is original andsignificant.

Example 7

Difference in the IL-12 production between reductive and oxidativemacrophages

When there are defects in differentiation, selection and functionalexpression steps of T cells, the host immune system becomes deficient.From this fact, it is considered that T cells play an central role inthe host immune system. Helper T cells (Th) which are one subset of Tcells produce lymphokines to regulate immunocytes or inflammatory cells.Recently, the following concept has been proposed. That is, Th isfurther classified into two types, Th1 and Th2 depending on the types ofthe lymphokines produced, and these cells have the differentimmunological functions (J. Immunol., vol. 136, pp. 2348, 1986). Thatis, Th1 produces IL-2 or IFNγ, and is a main cell to modulate cellularimmunity. Th2 produces IL-4, IL-5, IL-6 and IL-10, and is a main cell tomodulate humoral immunity. The homeostasis of the in vivo immunity ismaintained by the Th1/Th2 balance. Usually, when the Th1/Th2 balance isinclined to either of Th1 and Th2, the host correspond to correct theskewing and tend to maintain the homeostasis. However, it is consideredthat when the imbalance is not corrected for some reasons, immunologicaldiseases will occur. Th1 and Th2 are differentiated from the precursorof them, namely Th0. In the differentiation of Th0 to Th1, IL-12produced by MΦ is important (Immunology Today, vol. 335, p 14, 1993). Inthe differentiation of Th0 to Th2, IL-4 produced by NKT cells isimportant (J. Exp. Medicine, vol. 179, pp.1285,1994).

In the above-mentioned Example, it is clarified that the MΦ functiondiffers depending on the difference in the redox state of MΦ. Withrespect to MΦ, there are two types of MΦ, oxidative MΦ and reductive MΦbased on the difference in the GSH content, and these two distinctive MΦbehave differently in the NO or IL-6 production. The main producer ofIL-12, which induces differentiation of Th0 to Th1 and which is a keymolecule of controlling the Th1/Th2 balance, is considered to be MΦ.However, the detailed analysis has not yet been reported. In view of theclarification of attack mechanism of immunological diseases, it is quiteinteresting to know whether or not the IL-12 production is differentbetween oxidative MΦ and reductive MΦ. The present inventors have foundthat IL-12 is produced from only reductive MΦ, and that IL-4 consideredto control the Th1/Th2 balance like IL-12 acts on oxidative MΦ andreductive MΦ whereby the Th1/Th 2 balance is skewed to the Th2 side. Onthe basis of the findings which were obtained prior to the completion ofthe present invention, it is shown that the redox state of MΦ regulatesthe Th1/Th2 balance, and the usefulness of the present invention in thediagnosis of immunological diseases is described.

IL-12 is produced from reductive MΦO:

In Example 1, it was indicated that MΦ produced by injecting lentinan(LNT) intraperitoneally was reductive MΦ with the high GSH content andthat MΦ induced by injecting LPS intraperitoneally was oxidative MΦ withthe low GSH content. It was examined whether there is a difference inthe IL-12 production between LNT-induced MΦ and LPS-induced MΦ. Bystimulation with LPS and INFγ, the remarkable IL-12 production (1,312pg/ml) was observed in the LNT-induced MΦ. However, no IL-12 productionwas observed in LPS-induced MΦ and control resident MΦ (FIG. 4). Next,the same analysis was conducted using MΦ induced by intraperitoneallyinjecting substances for changing the GSH content in cells. With respectto MΦ induced by administering glutathione monoethyl ester (GSH-OEt), asubstance increasing the GSH content in cells and diethyl maleate (DEM),a substance decreasing the same, IL-12 (3,570 pg/ml) was produced onlyin MΦ derived from GSH-OEt administered mice through stimulation withLPS and IFNγ. These results show that IL-12 is produced only byreductive MΦ having the high GSH content in cells.

The IL-12 production from reductive MΦ is suppressed by decreasing theGSH content in cells:

As mentioned above, IL-12 was produced only in reductive MΦ having thehigh GSH content in cells. It was examined whether this production issuppressed by converting MΦ to oxidative MΦ. That is, it was analyzedwhether the IL-12 production is suppressed by exposing lentinan-inducedMΦ with DEM. As a result, it was clarified that the IL-12 production(828 pg/ml) from lentinan-induced MΦ is completely suppressed (0 pg/ml)with the addition of DEM. That is, it was suggested that reductiveglutathione in cells is deprived through DEM treatment and reductive MΦis converted to oxidative MΦ to suppress the IL-12 production.

IL-4 suppresses the IL-12 production by reductive MΦ:

IL-4 is a cytokine which acts on MΦ suppressively. IL-4 is considered tohave an opposite function to IL-12 in the Th1/Th2 balance as well.Accordingly, it was examined whether IL-4 acts suppressively on theIL-12 production by reductive MΦ. It was clarified that the IL-12production by LNT-induced MΦ and the IL-12 production by GSH-OEtadministered mouse MΦ are remarkably suppressed by the pretreatment withIL-4 (from 1, 580 pg/ml to 370 pg/ml and from 490 pg/ml to 258 pg/ml).That is, it was suggested that there is a possibility that IL-4 acts onMΦ to suppress the IL-12 production whereby the Th1/Th2 balance isskewed to the Th2 side. At this time, it was clarified from the imageanalysis by ACAS that IL-4 markedly decreases the content of reductiveglutathione in MΦ.

IL-4 suppresses the NO production and increases the IL-6 production:

Reductive MΦ increases the NO production by the IFNγ stimulation incomparison with oxidative MΦ, and rather suppresses the IL-6 production.IFNγ is known to be a cytokine produced from Th1 cells. What functionIL-4 shows in the NO production and the IL-6 production with IFNγ wasanalyzed using respective MΦ's. IFNγ NO production from MΦ pretreatedwith IL-4 was significantly suppressed in comparison with MΦ untreatedwith IL-4. Further, MΦ of which the GSH content in cells was increasedby the stimulation with GSH-OEt and MΦ of which the GSH content in cellswas decreased by the stimulation with DEM were pretreated with IL-4, andIFNγ thereafter and LPS stimulation was carried out to induce NOproduction. As a result, the NO production was remarkably suppressed inIL4-treated MΦ in comparison with IL4untreated MΦ in both subsets of MΦ.

Meanwhile, with respect to the IL-6 production, the production with IFNγwas markedly increased by pretreatment with IL-4 in resident MΦ,LPS-induced MΦ and LNT-20 induced MΦ.

Further, MΦ of which the. GSH content in cells was increased by thestimulation with GSH-OEt and MΦ of which the GSH content in cells wasdecreased by the stimulation of DEM were pretreated with IL-4, and IFNγwas exposed thereon to induce the IL-6 production. Consequently, theIL-6 production was increased in IL-4-treated MΦ's in comparison withIL4-untreated MΦ. These results revealed that IL-4 induces oxidativemacrophages by decreasing the content of reductive glutathione in cells,suppressing the NO production by the stimulation with IFNγ andincreasing the IL-6 production. This indicates that IL-4 suppresses theNO production by IFNγ, namely, Th1 type response, increases the IL-6production by IFNγ, and has an activity of enhancing Th2 type response.These findings scientifically prove the usefulness of theimmunomodulator according to the present invention.

Example 8

Enhancement of IL- 12 production with a combination of NAC orally takenin and IL-2 infusion

Two groups, namely, a group of 8-week-old DBA/2 female mice which werecaused to freely drink city water as in Example 6, and a group of thesame female mice which were caused to freely drink city water containing1 mg/ml of NAC, were prepared. Further, the two groups ofthe-above-mentioned mice to which human recombinant IL-2 in an amount of2 μg/0.5 ml/h was intraperitoneally administered twice a day, every twodays for two weeks were provided. On day 14, the IL-12 production fromMΦ was measured in the same manner as in Example 6.

Measurement of the GSH content in MΦ prepared:

The peritoneal cells were harvested from the mice which had undergonethe respective treatments, and the GSH content in cells was analyzed byACAS using an MCB reagent. In comparison with control mice (group causedto freely drink city water), the content of reductive glutathione wasmarkedly increased in the group caused to freely drink NAC-containingcity water and the IL-2 administration group, showing the image ofreductive MΦ.

The content of reductive glutathione was more increased in the groupwhich had undergone the combination of the free-drinking ofNAC-containing city water and the IL-2 administration than in any of thegroup of the free drinking of NAC-containing city water and the IL-2administration group. Thus, the effect brought forth by the combinationof the treatments in the induction of reductive MΦ was clearly observedin the ACAS image analysis. In the group which had undergone thecombination of the treatments, the increase in the content of reductiveglutathione was observed in all MΦ's (in contrast with the fact that theincrease in the content thereof in the group of the sole treatment wasobserved in from 40 to 50% of MΦ).

Function of MΦ produced from each group:

Peritoneal cells were harvested from four groups of the mice, andstimulated with LPS and IFNγ. Then, the NO production, the IL-6production and the IL-12 production were measured. Since the content ofreductive macrophage was increased in three groups of the soleadministration and the combined administration in comparison with thecontrol group, the amount of IL-6 in the macrophage culture supernatantwas decreased (relative to 1,240 pg/ml in control mice, 320 pg/ml in thegroup caused to freely drink NAC-containing city water, 520 pg/ml in theIL-2 administration group, and 67 pg/ml in the group which had undergonethe combination of the free-drinking of NAC containing city water andthe IL-2 administration). In consideration of the fact that IL-6 is amain cytokine inducing Th2, the combination of the NAC oral intake andthe injection of IL-2, the cytokine can control the Th1/Th2 balance morestrongly. The increase pattern of the NO production was inverselyrelated with the IL-6 production. With respect to the IL-12 production,the amount of IL-12 was 620 pg/ml in the group caused to freely drinkNAC-containing city water, 946 pg/ml in the IL-2 administration group,and 2,386 pg/ml in the group which had undergone the free drinking ofNAC-containing city water and the IL-2 administration in comparison with0 pg/ml in control mice. Thus, the remarkable effect was observed by thecombination of the treatments. It shows that the present inventionprovides the immunomodulator which is useful for the remarkableimprovement of the immunological diseases such as rheumatoid arthritisin combination with the cytokines, and is therefore original andsignificant.

Example 9

Induction of oxidative macrophages through administration of (NAC-OMe)₂,(NAC)₂, or acetylgliotoxin

Oxidative macrophages were induced by intraperitoneally administering 20μg/0.5 ml/h of (NAC-OMe)₂ or (NAC)₂, or 10 μg/0.5 ml/h ofacetylgliotoxin in DBA/2 mice on Day 1 and Day 2, and reductivemacrophages were induced by intraperitoneally administering 2 mg/0.5ml/h of NAC in DBA/2 mice on Day 1 and Day 2. This was clarified byharvesting peritoneal exudate cells 20 hours after the completion of theadministration, adhering the cells to the plastic surface, then reactingthe cells with 10 μM of monochlorobimane at 37° C. for 30 minutes, andanalyzing the reaction product with ACAS. The increase in the amounts ofoxidative macrophages can easily be determined visually from the factthat almost no reaction product with monochlorobimane is observed,namely, a gray or blue image is provided, and the increase in theamounts of reductive macrophages from the fact that a red or yellowimage is provided respectively. It was found that macrophages induced 20hours after subcutaneously administering 40 μg/0.1 ml/h ofdexamethasone, a typical steroid having an immunosuppressive activity aswell known on the back of a mouse on Day 1 and Day 2 gave almost a grayimage, namely, oxidative macrophages were strongly induced. Meanwhile,the same examination was conducted by harvesting peritoneal exudatecells after 20 hours of the administration of 2 mg of N-acetylcystine(NAC). Consequently, a red or yellow image was obtained, and it wasidentified that reductive macrophages were induced. Similarly, theinduction of oxidative macrophages was confirmed on ACAS, byintraperitoneally administering cystine derivatives, i.e.,N,N′-diacetylcystine nitroxybutyl ester, N,N′-diacetylcystine dimethylester (NAC-OMe)2), N,N′-diacetylcystine diisopropyl ester (NAC-OiPr)₂)and N,N′-di-L-alanylcystine dimethyl ester (NAlaC-OMe)₂) (administering20 μg/0.5 ml/h each, twice a week for three weeks, 9-11 weeks of age).

<Production of NO and IL-6 from macrophages induced throughadministration of (NAC-OMe)₂, (NAC)₂ or acetylgliotoxin>

Thus, the peritoneal exudate adherent cells were incubated in thefollowing manner, and the amounts of NO and IL-12 produced in theculture supernatant were measured. With respect to the amount of IL-6,the amount of IL-6 produced spontaneously in the absence of a stimulatorwas measured.

(1) Materials

Cells: The peritoneal exudate adherent cells obtained by thestimulation, namely, the macrophages were added to a 96-well microplatein an amount of 1×10⁵ cells/200 μl each.

Medium: Phenol red-free RPMI 1640: 200 μl/well

LPS: Lipopolysaccharide (made by Sigma Co.) (origin: E. coli) 100 ng/ml

IFNγ: 100 units/ml

(2) Measuring method

Harvest of peritoneal MΦ:

Peritoneal cells were harvested by injecting 5 ml of a phenol red-freeDMEM medium (supplied by Nikken Seibutsusha) ice-cooled into aperitoneal cavity of a mouse which had been put to sacrificial deathwith ether using an injection cylinder fitted with a 22-gauge needle,squeezing the same and pulling out the medium.

Determination of the amount of IL-6:

Conducted as in Example 2.

Measurement of a concentration of NO₂:

Conducted as in Example 2.

Determination of GSH in cells with ACAS:

Conducted as in Example 2.

Determination of an amount of IL-12:

Conducted as in Example 2.

Results:

The effects of inhibiting production of NO, IL-6 and IL-12 frommacrophages are shown in Table 1.

TABLE 1 (Amount of produced relative to that of control group, %) SampleNO IL-6 IL-12 (NAC—OMe)₂ 12 320 11 (NAC)₂ 18 340 15 Acetylgliotoxin 22450 14 NAC 172   42 920  Dexamethasone 45 1020   5

As is apparent from Table 1, the amounts of inflammatory cytokines IL-6,NO and IL-12 produced are changed with the oxidative macrophages inducedthrough the administration of (NAC-OMe)₂, (NAC)₂ and acetylgliotoxin.That is, with the oxidative macrophages obtained through theadministration of the agent, the IL-6 production is increased, and theproduction of NO, which induces tissue injury, and the production ofIL-12, which increases the cellular immunity, are both decreased. Thiseffect is the same as, or higher than, that of dexamethasone, a typicalsteroid-type immunosuppressant. On the contrary, with the reductivemacrophages induced by N-acetylcysteine (NAC), the NO production and theIL-12 production are increased, and the IL-6 production is reduced.

Example 10

Effect of inhibiting delayed type hypersensivity reaction to ovalbumin

20 g/0.5 ml/h of (NAC-OMe)₂, 20 g/0.5 ml/h of (NAC)₂, 2 mg/0.5 ml/h ofNAC and 30 g/0.5 ml/h (Day 1) of dexamethasone were continuouslyadministered from Day 1 to Day 5 as in Example 9. With respect to anantigen, 100 μl of a suspension of ovalbumin and Complete H37Ra Adjuvant(Difco) at a ratio of 1:1 (containing 250 μg of ovalbumin) wereadministered subcutaneously to the back as a sensitization antigen onDay 2 and to the left ear as an induction antigen on Day 8. After 24hours, the thickness on the left ear was compared with that of the rightear.

The results of the effect of inhibiting the delayed typehypersensitivity reaction to the ovalbumin antigen are shown in Table 2.The administration of (NAC-OMe)₂ and (NAC)₂ remarkably inhibited thedelayed type hypersensitivity reaction to the ovalbumin antigen. Thisreveals that the administration of these substances inhibits thecellular immunity.

TABLE 2 Increase in the ear Sample thickness (mm) Control group 15.1(NAC—OMe)₂ 9.75 (NAC)₂ 9.88 NAC 16.5 Dexamethasone 4.75

Example 11

Function of macrophages in an animal in which a gene is knocked out

For clarifying a mechanism of chronic or advanced inflammation, it isimportant to analyze, at a molecular level, why there is a difference inproduction of an inflammatory mediator or cytokine between oxidative MΦand reductive MΦ. Generally, extracellular stimulation (ligand) istransferred into cells through a receptor present on cell surfaces.Various kinases are activated with a signal from a receptor, andtranscriptional factors are also activated, translocated to the nucleus,and bound to target genes for expression. According to the recentstudies, it is being clarified that a redox system in cells participatesin activation of transcriptional factors, shifting of the same into thenucleus and binding of the same to genes (refer to Annual Rev.Immunology, vol. 8, pp 453-457, and Embo J., 10, pp. 2247-2251, 1991).At present, it is unknown how a redox system in cells participates in agene expression system through a receptor in MΦ. As one approach f orclarifying it, MΦ's were produced from knock out mice deficient in amolecule participating in a signal translocation system from a receptor,and the function of the redox system was analyzed. Specifically, acommon γ chain (γc) used in common as a receptor constituting moleculeof IL-2, IL-4, IL-7, IL-9 and IL-15 was used as a target molecule.

Cytokine and stimulator:

As mouse IFNγ, a recombinant supplied by Genzyme was used. As human IL-2and human IL-6, recombinants supplied by Ajinomoto Co. Inc. were used.As human IL-12, a recombinant supplied by Pharmingen was used.

As LPS, a substance derived from E. coli 055:B5 supplied by Difco wasused. As lentinan, a preparation produced by Ajinomoto Co. Inc. wasused.

Mice used:

γc knock out mice were obtained from Professor Sugamura, TohokuUniversity Medical School.

As wild mice used for mating and as a control, C57BL/6 obtained from CRJwas used.

Measurement of the GSH content in MΦ produced from knock out mice:

Peritoneal cells were harvested from γc knock out mice, and the GSHcontent in cells was analyzed by ACAS using an MCB reagent. The contentof reductive glutathione in MΦ derived from γc knock out mice wasremarkably decreased in comparison with that in control mice (C57BL/6).

Function of MΦ harvested from knock out mice:

Peritoneal cells were produced from wild mice (C57BL/6) and γc knock outmice, and stimulated with IPS, IL-2, IFNγ and a combination thereof TheNO production, the IL-6 production and the IL-12 production weremeasured. Almost no NO production was observed in any mice in theabsence of stimulation. In the stimulation with the combination of LPSand IFNγ, the decreased NO production in γc knock out mice derived MΦwas observed to less than half that in control mice. Further, the IL-6production was analyzed. In the LPS stimulation, an increase in the IL-6production was observed in γc knock out mice (962 pg/ml relative to 81pg/ml of a control). In the IFNγ stimulation, an increase in the IL-6production was observed in γc knock out mice. Still further, the IL-12production with the LPS stimulation and the IFNγ stimulation wasexamined. No production was observed at all in any mice. This provesthat in the gene knock out mice used herein, the amount of the oxidativemacrophages is increased to increase the humoral immunity or theallergic reaction mainly caused by Th2 and to decrease the cellularimmunity supported by TH1. In the animal disease models, it is clearlyshown that the invention is original and significant in the diagnosis ofimmunological diseases.

Example 12

Spontaneous progression of inflammatory bowel disease in γc knock outmice

In wild-type normal littermates of γc knock out mice (genetic phenotypes+/+, +/Y), no inflammatory bowel disease is observed at all underordinary SPF breeding conditions. However, in γc knock out mice (geneticphenotypes, −/−, −/+ and −/Y), inflammatory bowel diseases occur very often. With respect to homo-knock out mice with −/− and −/Y phenotypes,approximately 70% thereof are spontaneously accompanied withinflammatory bowel diseases within 4 months. Also with respect to micewith +/− phenotype, approximately 60% thereof are spontaneouslyaccompanied therewith within 6 months. That is, intestinal shortening,bloody stool, diarrhea, loose passage, anal prolapse and colonalhypertrophy are observed.

With respect to an intestinal inflammation model, the oral intake ofdextran sulfate is well known. However, an inflammatory image of γcknock out mice is by far similar to that observed in inflammatory boweldiseases of humans. In the histochemical analysis using pathologicsamples, the following fact was clarified.

Analysis of pathologic specimens was conducted with respect toformalin-immobilized HE dyed specimens of the large intestinecorresponding to the colon in a portion which was from 2 to 3 cm fromthe anus. The evaluation was conducted with respect to five points, 1)longitudinal spreading of inflammatory cell invasion in a direction of amucous epithelium—lamina propria mucosae—lamina muscularismucosae—submucous layer—internal circular layers of musculartunics—external longitudinal layers of muscular tunics—serous membrane,2) lateral spreading thereof, 3) types of invaded cells, 4) degree ofneovascularization and 5) hypertrophy of a submucous layer. Invasion ofinflammatory cells was scarcely observed in +/Y normal samples,wild-type littermates of γc knock out mice, and the mucous structure wasretained almost intact in both goblet cells and mucous epithelium cells.In the group of the free drinking of 1% dextran sulfate water, drop ofneutrophils and inflammatory cells in the gland cavity was observed, anddegeneration and disappearance of goblet cells and metaplasia anddegeneration of the mucous epithelium were identified at high levels.Invasion of lymphocytes, MΦ and neutrophils, neovascularization andvasodilation occurred at high levels. It was rated as grade 4.

In untreated samples of γc knock out mice with genotypes, the mucousepithelium was nearly intact, but hyperplasia was observed. Drop ofinflammatory cells into the gland cavity did not occur. No edema wasobserved in the submucous layer, nor did invasion in the muscular tunicsand the submucous layer occur. Invasion of MΦ and lymphocytes wasobserved only in the lamina propria mucosae, and unlike dextransulfate-induced models, invasion of neutrophils was not observed. It wasdifferent from a mere acute inflammation image, and close to a humaninflammatory bowel disease pathology image.

In untreated samples of γc knock out mice −/Y, the mucous epithelium was2 or 3 times that of wild-type mice, and invasion of inflammatory cellswas observed only in the lamina propria mucosae as a local cluster. Themucosa in contact with the cluster in the untreated samples of γc knockout mice +/− was close to the normal one compared with that of mice −/−and −/Y. The invasion cluster of inflammatory cells was observed in thebottom of the lamina propria mucosae only at a low level.

Accordingly, the intestinal inflammation of γc knock out mice with −/−and −/Y genotypes is similar to that of humans, and the dextransulfate-induced model is considered different in mechanism from the γcknock out mouse spontaneous inflammatory bowel disease model.

Example 13

Effect of inhibiting spontaneous inflammatory bowel diseases in γc knockout mice

On the basis of the findings mentioned above, the effect of inhibitingspontaneous progression of inflammatory bowel disease in γc knock outmice was examined with respect to human Crohn disease and ulcerativecolitis models. Saline, (NAC-OMe)₂ or (NAC)₂ (20 μg/h) was administeredtwice a week, 5 times in total, to each of 6 γc knock out male mice with+/Y genotype and 6 γc knock out male mice with −/Y genotype which wereinclined to have oxidative MΦ. It was examined whether or not theintestinal inflammation spontaneously progressed in −/Y was inhibited.In case of (NAC-OMe)₂, on Day 14 of the administration, the occurrencerate of the non-administration −/Y group and the administration −/Ygroup was 83% to 25%; on Day 31, the occurrence rate was 83% to 25%, andthe survival rate was 33% to 100%; and on Day 45, the occurrence ratewas 100% to 25%, and the survival rate was 33% to 75%. In case of(NAC)₂, it was 30, 40, 100, 40, 75% respectively. Thus, it wasidentified that the substance used in the invention clearly exhibitedthe effect to prolong survival of life and the effect of preventing thedisease progression in the intestinal inflammation spontaneouslyinduced. By the way, in the non-administration +/Y group and theadministration +/Y group, the disease progression did not occur, andthere was no difference therebetween.

Example 14

Effect of inhibiting dextran sulfate-induced inflammatory bowel diseasein γc knock out mice

To 3-week-old γc knock out mice with +/− genotypes were administered 20μg/h of (NAC-OMe)₂ three times a week for 2 weeks. On day 16, the freedrinking of 1% dextran sulfate water was started. On Day 30, theoccurrence rate of the inflammatory bowel disease was 100% in thenon-administration group and 16.6% in the administration group, and theeffect of the administration of this agent was observed. Further, thesurvival rate was 57% in the non-administration group and 100% in theadministration group. Thus, the remarkable effect was observed. Withrespect to the +/+ mice, the occurrence rate was 60% in theadministration group and 80% in the non-administration group. Therefore,the effect of the administration was also observed. The γ knock out miceand their wild-type male mice (+/Y -and −/Y) and female mice (+/+, +/−and −/−) were caused to freely drink water containing 1% dextran sulfateas a inflammatory bowel disease model. The female mice showed aresistance, and the order of the resistance was found to be −/Y and +/Yin male mice, and −/−, +/− and +/+ in female mice. After the start-up ofthe administration of dextran sulfate, the survival rate was 80, 0, 100,100 and 40% on Day 13, showing that the mice mainly having oxidative MΦwere more resistant to the dextran sulfate-induced gastrointestinalinflammation. This indirectly proved the invention to be reasonable.

Example 15

Functions of macrophages in synovial cells from rheumatoid arthritispatients

So as to elucidate the mechanism of chronic exacerbation ofinflammation, it is significant to analyze at a molecular level why thedifference in the generation of an inflammatory mediators and cytokinesemerges between oxidized MΦ and reduced MΦ. Generally, extracellularstimuli (ligands triggering) are intracellularly transmitted viaspecific receptors present on cell surface. Intracellular signals fromthe receptors activate various kinases and additionally activatetranscriptional factors, so that the transcriptional factors translocateinto nuclei and bind to the enhancer/promoter sequence of a target genetherein, leading to the expression thereof. Recent research works areunder way of elucidating that intracellular redox systems areparticipating in activation, nuclear translocation and DNA binding oftranscriptional factors [ANNUAL REV. IMMUNOLOGY, Vol. 8,453-475 (1990);EMBO J., 10, 2247-2251 (1991)]. Up to now, however, it has not yet beenelucidated how the intracellular redox systems are involved in thereceptor-mediated gene expression systems of inflammatory mediators orcytokines in macrophages.

Cytokine and stimulator:

As mouse IFNγ, a recombinant supplied by Genzyme was used. As human IL-2and human IL-6, recombinants supplied by Ajinomoto Co. Inc. were used.As human IL-12, a recombinant supplied by Pharmingen was used.

As LPS, a substance derived from E. coli 055:B5 supplied by Difco wasused. As lentinan, a preparation produced by Ajinomoto Co. Inc. wasused.

Joint-derived macrophages:

Synovial tissue was aseptically sampled from tissues resected duringjoint surgery. The sampled synovial tissue was rinsed in phosphatebuffer, from which the surface was peeled off in a petri dish containingthe same phosphate buffer. Then, the resulting synovial tissue was cutinto pieces with a pair of scissors. To the pieces were added 2%hyaluronidase (manufactured by Sigma), 0.2% DNase (derived from bovinepancreas) and 5% collagenase, for enzymatic treatment at 37° C. for 2hours. After discarding the resulting debris, a cell fraction recoveredby centrifugation was adjusted to 5×10⁵ cells/ml in a phenol red-freeDMEM culture medium supplemented with 1 0%FCS (manufactured by NikkenBiology Co.). For 3 hours, the cells were allowed to adhere to theplastic surface. Plastic surface adherent macrophage-like cellsharvested with a rubber policeman were then subjected to rinsing incombination with centrifugation three times using the culture mediumunder ice cooling. The resulting cells were designated as synovialtissue-derived macrophages and were then subjected to the followingexperiments.

Determination of the amount of IL-6:

A stimulator was added to 1×10⁶ MΦ, and the incubation was conducted at37° C. for 2 days in a CO₂ incubator. After centrifugation, the culturesupernatant was collected.

The amount of IL-6 was determined using IL- 6 dependent mouse hybridomaMH60 cells (J. Eur. Immunol., vol. 18, p. 951, 1988). One hundredmicroliters of the culture supernatant were added to 100 μl of the MH60cell suspension adjusted to 1×10⁵ cells/ml in a 10% FCS-containing RPMImedium, and the mixed solution was incubated at 37° C. for 2 days in aC0₂ incubator. Subsequently, 10 μl of MTT (supplied by Sigma Co.)solution adjusted to a concentration of 5 mg/ml in the same medium wereadded thereto, and the reaction was conducted at 37° C. for 5 hours.After the completion of the reaction, the centrifugation was conducted.The supernatant (160 μl) was removed, and 100 μl of a mixture ofhydrochloric acid and propanol were added to the residue. The suspensionwas conducted using a pipetman to dissolve the cells. Immediately afterthe dissolution, an absorbance of 570 nm was measured with animmunometer (supplied by Bio-Rad).

Measurement of a concentration of NO₂:

A stimulator was added to 1×10⁵ MΦ, and the incubation was conducted at37° C. for 2 days in a CO₂ incubator. After the completion of thecentrifugation, the culture supernatant was collected.

One hundred microliters of a Griess-Romijn reagent (supplied by WacoPure Chemical Industries, Ltd.) adjusted to a concentration of 50 mg/mlin distilled water were added to 100 μl of the culture supernatant, andthe reaction was conducted at room temperature for 15 minutes. After thecompletion of the reaction, an absorbance of 540 nm was measured. NaNO₂was used as a standard.

Determination of GSH in cells with ACAS:

Three-hundred microliters of a cell suspension adjusted to aconcentration of 3×10⁵ cells/ml in an RPMI 1640 medium (phenol red-free)were charged into a chambered coverglass (#136439, supplied by Nunc),and incubated at 37° C. for 2 hours using a CO₂ incubator. The culturesolution was washed with the same medium, and 300 μl of monochlorobimane(supplied by Molecular Plobe) adjusted to 10 μM in the same medium wereadded thereto. The mixture was charged into a CO₂ incubator of 37° C.,and the reaction was conducted for 30 minutes. The fluorescent intensitywas measured with ACAS. In ACAS, a UV laser was used.

Determination of an amount of IL-12:

The amount of IL-12 was determined through bioassay using cells of humanT cell strain 2D6 (J. Leukocyte Biology, vol. 61, p. 346, 1997).

2D6 cells which had been incubated in an RPMI 1640 medium containing 500pg/ml of recombinant human IL-12, 50 μM of 2-mercaptoethanol and 10% FCS(fetal calf serum) were moved to a tube, and centrifugally washed threetimes with the above-mentioned medium without IL-12 and cell density wasadjusted to 1×10⁵/ml. The cell suspension was added in an amount of 100μl each to a 96-well flat bottom plate containing a sample seriallydiluted in advance with an RPMI 1640 medium containing 50 μM of2-mercaptoethanol and 10% FCS in an amount of 100 μl each. Subsequently,the mixture was charged into a 5% CO₂ incubator of 37° C., and incubatedfor 48 hours. For final 6 hours, ³H-TdR was pulsed (a substance adjustedto 370 kBq/ml in an RPMI 1640 medium containing 50 μM of2-mercaptoethanol and 10% FCS was added in an amount of 50 μl each). Thecells were harvested, and the radioactivity was measured using a counter(Matrix 96, supplied by Packard).

Example 16

GSH concentration in MΦ prepared from synovial tissue from rheumatoidarthritis patients

By ACAS with an MCB reagent, the GSH concentration in themacrophage-like cells prepared by the aforementioned method wasdetermined. The macrophages collected were suspended and adjusted in aphenol red-free RPMI 1640 culture medium supplemented with 10% fetalcalf serum to 3×10⁶ cells/ml; the suspension was divided in 100-μlportions on Lab-Tek Chamber Slide (#136439; manufactured by NUNC, CO.),for culturing in 5% CO₂ at 37° C. for 3 hours; after discarding thenonadherent cells, the serum-free culture medium of 200 μl was fed tothe resulting culture, followed by addition of MCB (abbreviation formonochlorobimane) to a final concentration of 10 μM, for reaction for 30minutes; and the amount of intracellular GSH was assayed by imaginganalysis with an ACAS system (manufactured by Meridian Co.).

Results:

Reduced glutathione was quantitatively assayed by the ACAS method. Inthe macrophages derived from tissues of rheumatoid arthritis patients atactive stage, compared with the macrophages derived from tissues ofpatients with osteoarthritis, the relative ratio of oxidativemacrophages, namely macrophages with the reduced amount of glutathione,was increased. Because of the increase of oxidative macrophages, IL-6 inthe culture supernatant of the macrophages was prominently increased. Itis thus indicated that the identified redox state of macrophages on thebasis of the glutathione content assay can suggest the pathologicalstate and immune function of a rheumatoid arthritis patient in a simpleand appropriate manner, with no need of determination of numerousfunctional parameters of macrophages. For the administration of theinventive anti-rheumatoid agent, thus, the pathological state and immunefunction of a patient can be examined and diagnosed according to theaforementioned macrophage classification method.

Function of MΦ prepared from joint cavities of rheumatoid arthritispatients:

Macrophages were prepared from synovial tissues of patients withosteoarthritis and rheumatoid arthritis patients at active stage by thesame method as described above, which were then stimulated with LPS,IFNγ and a combination thereof for assaying the abilities of NO andIL-12 generation. With no stimulation, almost no IL-12 generation wasobserved in the macrophages from any of the origins; by stimulation witha combination of LPS and IFNγ, no such generation was observed therein.The level of NO generation in the macrophages from patients withactive-stage rheumatoid arthritis was decreased ½-{fraction (1/4+L)}-fold the level in the control, which indicates that oxidativemacrophages predominant in the rheumatoid arthritis patients at activestage potentiate humoral immune response driven by Th2, while cellularimmune response for which Th1 is responsible is declined in thesepatients. The findings described above suggest that the pathologicalstate of rheumatoid arthritis patients can be diagnosed by assaying theGSH content in macrophages, which is beneficial to determine the way forthe administration and application of the inventive anti-rheumatoidagent. Thus, the creativeness and significance of the invention can thusbe exemplified.

Example 17

Modification of macrophage redox function with addition of chemicalagents to culture system of macrophage-like cells from tissues ofrheumatoid arthritis patients

By the same manner as in Example 3, macrophage-like cells were collectedfrom tissues of rheumatoid arthritis patients, and were then suspendedand adjusted in a phenol red-free RPMI 1640 culture medium supplementedwith 10% fetal calf serum to 3×10⁶ cells/ml. The suspension was dividedin 100-μl portions on Lab-Tek Chamber Slide (#136439; manufactured byNUNC, CO.), for culturing in 5% CO₂ at 37° C. for 3 hours; afterdiscarding the nonadherent cells, the 200-μl serum-free culture mediumfurther containing any of the following chemical agents was fed to theresulting culture, for reaction for 3 hours; and then, the resultingculture was rinsed three times, followed by addition of the serum-freeculture medium of 200 μl and addition of monochlorobimane to a finalconcentration of 10 μM, for reaction for 30 minutes. The amount ofintracellular GSH was assayed by imaging analysis with an ACAS system(manufactured by Meridian Co.).

Results:

Reduced glutathione was quantitatively assayed by the ACAS method. Inthe glutathione ethyl ester (2 mM)-added group compared with the controlphysiological saline-added group, the relative ratio of oxidativemacrophages, namely macrophages with the reduced amount of intracellularglutathione, was decreased. Because of the increase of reductivemacrophages, IL-6 in the culture supernatant of the macrophages wasprominently decreased. The same action was also observed in groups withaddition of γ-glutamylcysteine dimethyl ester, N-acetylcysteinenitroxybutyl ester, glutathione monoethyl ester, glutathionenitroxybutyl ester and glutathione diethyl ester (all added at 2 mM).The induction of oxidative macrophage with the reduced amount ofintracellular glutathione was confirmed in groups with addition ofcystine derivatives such as N,N′-diacetylcystine nitroxybutyl ester, N,N′-diacetylcystine dimethyl ester [(NAC-OMe)₂], N,N′-diacetylcystinediisopropyl ester [(NAC-OiPr)₂], and N,N′-di-L-alanylcystine dimethylester [(NAlaC-OMe)₂] (all added at 2 μM).

Example 18

Effects of chemical agents administered to rats with adjuvant-inducedarthritis (AA)

Adjuvant-induced arthritis was triggered in Lewis rats in a conventionalmanner; and the effects of chemical agents were examined in the animals.Adjuvant-induced arthritis is well known as an experimental arthritisinducible in rats [Taurog et al., Meth. Enzymol. 162, 339-355 (1988)].It is suggested that clinical efficacy of non-steroidal acidanti-inflammatory agents and immunosuppressive agents such ascyclosporin, cyclophosphamide and methotrexate as antirheumatoid agentscan be reflected in the model. An adjuvant is administered at 1 mg/0.1μml per rate at a site of caudal root. The edema reaches its peak about2 to 3 weeks after the administration of the adjuvant. The edema istriggered by inflammatory infiltration of monocytes. Swollen joints aremeasured with a micrometer every day; comparing the diameters of thejoints prior to and after adjuvant administration and after initiationof the treatment with chemical agents with the diameters of the jointsin the control group, the effects of the chemical agents were expressedas the ratio in % of joint swelling in the experimental groups to jointswelling in the control group. The effects were determined on day 15after adjuvant administration. The chemical agents were given, startingthe day next to the day of adjuvant administration, and every 3 daysthereafter. The results are shown in Table 3.

TABLE 3 Ratio in % of joint swelling in experimental Chemical Agentsgroups to joint swelling in control group Physiological saline 100 (NAC—OMe)₂ 50 (NAC)₂ 96 NAC 132  Lentinan 52

The results based on the experiments of drug efficacy apparentlyindicate that adjuvant-induced arthritis can be suppressed by theadministration of a substance inducing oxidative macrophages. A singledose is 1 mg/kg for (NAC)₂ and (NAC-OMe)₂; 100 mg/kg for NAC; and 0.1mg/kg for lentinan.

Example 19

Effects of administration of chemical agents, including oraladministration thereof

Adjuvant-induced arthritis was induced in Lewis rats in a conventionalmanner. In the same manner as in Example 11, the effects of chemicalagents were examined in these rats. The conditions were the same asdescribed above. The results are shown in Table 4.

TABLE 4 Ratio in % of joint swelling in experimental groups to jointChemical Agents swelling in control group Physiological saline 100Intravenous (NAC—OMe)₂  42 Intravenous (NAlaC—OMe)₂  45 Intravenous(NAC)₂  95 Oral NAC 142 Intraperitoneal γ-glutamylcysteine 120 dimethylester Intraperitoneal glutathione diethyl ester 132 Intravenous lentinan 50

The doses were similar to those in Example 11; the doses of (AlaC-OMe)₂,γ-glutamylcysteine dimethyl ester, and glutathione diethyl ester wereall 100 mg/kg.

The above results apparently indicate that compounds represented by thestructural formula 1, including N,N′-diacetylcystine [(NAC)₂],N,N′-dipropylcystine [(NPC)2], N,N′-diacetylcystine dimethyl ester[(NAC-OMe)₂], N,N′-diacetylcystine diisopropyl ester [(NAC-OiPr)₂] andN,N′-di-L-alanylcystine dimethyl ester [(NAlaC-OMe)₂], and nitroxybutylesters thereof and exerting actions to decrease the content of reducedglutathione in macrophages, suppress delayed type hypersensitivityreactions, and suppress the generation of IFNγ or IL-12, arepharmacologically effective when administered to animals withadjuvant-induced arthritis.

Even when the compounds were administered 3 times per week for 3 weeks,the effects on the suppression of joint swelling were also observed inmodel mice with the spontaneous onset of rheumatoid arthritis. In themodel mice, swelling of foreleg finger bone joints was started on age 2months, together with pannus formation in the synovial tissue of thejoints under observation; by the dosing of the compounds with actions todecrease the content of reduced glutathione in macrophages, suppressdelayed type hypersensitivity reactions, and suppress the generation ofIFNγ or IL-12 to the animals, however, the joint score 4.5 months laterwas suppressed to 40 to 50% of the score in the physiologicalsaline-dosed control group. Alternatively, it is revealed that jointswelling is enhanced by reductive macrophage-inducing substancesincluding glutathione precursors such as γ-glutamylcysteine,γ-glutamylcysteine dimethyl ester, and N-acetylcysteine nitroxybutylester, and glutathione derivatives such as glutathione monoester,glutathione nitroxybutyl ester and glutathione diester.

Example 20

Functions of macrophages in NOD mice with spontaneous diabetes mellitus

So as to elucidate the mechanism of chronic exacerbation ofinflammation, it is significant to analyze at a molecular level why thedifference in the generation of an inflammatory mediators and cytokinesemerges between oxidized MΦ and reduced MΦ. Generally, extracellularstimuli (ligands triggering) are intracellularly transmitted viaspecific receptors present on cell surf ace. Intracellular signals fromthe receptors activate various kinases and additionally activatetranscriptional factors, so that the transcriptional factors translocateinto nuclei and bind to the enhancer/promoter sequence of a target genetherein, leading to the expression thereof. Recent research works areunder way of elucidating that intracellular redox systems areparticipating in activation, nuclear translocation and DNA binding oftranscriptional factors [ANNUAL REV. IMMUNOLOGY, Vol. 8, 453-475 (1990);EMBO J., 10, 2247-2251 (1991)]. Up to now, however, it has not yet beenelucidated how the intracellular redox systems are involved in thereceptor-mediated gene expression systems of inflammatory mediators orcytokines in macrophages.

Cytokine and stimulator:

As mouse IFNγ, a recombinant supplied by Genzyme was used.

As human IL-2 and human IL-6, recombinants supplied by Ajinomoto Co.Inc. were used. As human IL-12, a recombinant supplied by Pharmingen wasused.

As LPS, a substance derived from E. coli 055:B5 supplied by Difco wasused. As lentinan, a preparation produced by Ajinomoto Co. Inc. wasused.

Mice used:

NOD mice as model animals of insulin-dependent diabetes mellitus werepurchased from Nippon Clair, Co. Among them, female mice were mainlyused. Wild-type ICR mice purchased from Nippon Charles River, Co. (CRJ)were used as controls. Insulin-non-dependent diabetes mellitus-diseasedanimals were db/db mice purchased from Nippon Clair, Co.

Harvest of peritoneal MΦ:

Peritoneal cells were harvested by injecting 5 ml of a phenol red-freeDMEM medium (supplied by Nikken Seibutsusha) ice-cooled into aperitoneal cavity of a mouse which had been put to sacrificial deathwith ether using an injection cylinder fitted with a 22-gauge needle,squeezing the same and pulling out the medium.

Determination of the amount of IL-6:

Measurement of a concentration of NO₂:

Determination of GSH in cells with ACAS:

Determination of an amount of IL-12:

These were conducted in the same manner as in Example 15.

Measurement of the GSH content in MΦ produced from NOD mice:

Peritoneal cells were prepared according to the method stated above, andthe GSH content in cells was analyzed by ACAS using an MCB reagent. Thecontent of reductive glutathione was clearly decreased in the NOD miceat the age of 3-5 weeks, and was clearly increased in those accompaniedwith the disease (diabetes), as compared with control mice.

Function of MΦ produced from NOD mice:

Peritoneal cells were prepared from wild mice and NOD mice, andstimulated with LPS, IL-2, IFNγ and a combination thereof. The NOproduction and the IL-12 production were measured. Almost no IL-12production was observed in any mice derived MΦ in the absence ofstimulation. In the stimulation with the combination of LPS and IFNγ, noIL-12 production was observed in the NOD mice at the age of 3-5 weeks,but the IL-12 production was observed in those accompanied with thedisease. The NO production was decreased to from one third to one fourthin the NOD mice at the age of 3-5 weeks, but was increased to from twoto three times in those accompanied with the disease, as compared withcontrol mice. This proves that in the NOD mice used herein as a model ofspontaneous diabetic mellitus, the oxidative macrophages are predominantat the stage of infiltration of inflammatory cells in pancreatic islet,thereby increasing the humoral immune response mainly caused by Th2 anddecreasing the cellular immune response supported by Th1. On the otherhand, reductive macrophages are predominant at the stage of occurrenceof diabetes and deficient insulin secretion by destruction of islets ofLangerhans, thereby increasing the cellular immune response mainlycaused by Th1 and decreasing the humoral immune response supported byTh2. In the animal disease models, it is clearly shown that theanti-diabetes agent provided by the present invention and the diagnosisof diseases required for applying the agent is original and significant.

Example 21

Redox states of macrophage in NOD mice

Method:

Macrophages were sampled from the abdominal cavities of the NOD mice andthe control mice, to identify whether the macrophages were of anoxidative form or a reductive form. After physiological saline of 5 mlwas administered intraperitoneally into these mice, intraperitonealmacrophages were collected, which were then suspended and adjusted in aphenol red-free RPMI 1640 culture medium supplemented with 10% fetalcalf serum to 3×10⁶ cells/ml. The suspension was divided in 100-μlportions on Lab-Tek Chamber Slide (#136439; manufactured by NUNC, Co.),for culturing in 5% CO₂ at 37° C. for 3 hours; after discarding thenonadherent cells, the serum-free culture medium of 200 μl was fed tothe resulting culture, followed by addition of MCB (abbreviation formonochlorobimane) to a final concentration of 10 μM f or reaction f or30 minutes; and based on UV absorption, GSH was assayed by imaginganalysis with an ACAS system (manufactured by Meridien Co.).

Results:

Reduced type of glutathione was quantitatively assayed by the ACASmethod. In the NOD mice of age 3 to 5 weeks compared with the controlmice, the relative ratio of oxidative macrophages, namely macrophageswith the reduced amount of intracellular glutathione, was increased.Because of the increase of oxidative macrophages, IL-6 in the culturesupernatant of the macrophages was prominently increased (at 430 pg/mlvs at 120 pg/ml in the control mice). In the mice with the onset of thediabetes, macrophages with increased amount of intracellular reducedglutathione, namely reductive macrophages, were relatively increased. Itis thus indicated that the identified redox state of macrophages on thebasis of the glutathione content assay can suggest the pathologicalconditions and immune function of a diabetic patient in a simple andappropriate manner, with no need of determination of numerous functionalparameters of macrophages. For the administration of the inventiveanti-diabetes mellitus agent, thus, the pathological conditions andimmunological state of a diabetic patient can be examined and diagnosedaccording to the aforementioned macrophage classification method.

Example 22

Induction of reductive macrophages by chemical agents administered toNOD mice of age 3 to 5 weeks

Through a probe, glutathione ethyl ester was orally given at 1 mg/0.5ml/h/day to the NOD mice of age 4 weeks on alternate days, in total fivetimes. By the same method as in Example 3, intraperitoneal cells werecollected from the mice to sample intraperitoneal macrophages, whichwere then suspended and adjusted in a phenol red-free RPMI 1640 culturemedium supplemented with 10% fetal calf serum to 3×10⁶ cells/ml. Thesuspension was divided in 100-μl portions on Lab-Tek Chamber Slide(#136439; manufactured by NUNC, Co.), for culturing in 5% CO₂ at 37° C.for 3 hours; after discarding the nonadherent cells, the serum-freeculture medium of 200 μl was added to the resulting culture, followed byaddition of MCB (abbreviation for Monochlorobimane) to a finalconcentration of 10 μM for reaction for 30 minutes; and based on UVabsorption, intracellular GSH was assayed by imaging analysis with anACAS system (manufactured by Meridian Co.).

Results:

Reduced glutathione was quantitatively assayed by the ACAS method. Inthe NOD mice dosed with glutathione ethyl ester, compared with thecontrol NOD mice administered with physiological saline, the relativeratio of oxidative macrophages, namely macrophages with the decrease inthe content of reduced glutathione, was decreased. Because of theincrease of reductive macrophages, IL-6 in the culture supernatant ofthe macrophages was prominently decreased (at 460 pg/ml in the NOD micevs at 3800 pg/ml in the control group). It is thus indicated that theredox state of macrophages can be improved by glutathione ethyl esterorally administered. The same action is also observed forintraperitoneal dosing of γ-glutamylcysteine dimethyl ester (2 mg/0.5ml/animal, administered on alternate days, in total five times, startingfrom the age 4 weeks), intraperitoneal dosing of N-acetylcysteinenitroxybutyl ester (0.5 mg/0.5 ml/animal, administered on alternate daysin total five times, starting from the age 4 weeks), oral dosing thereof(1 mg/0.5 ml/animal, administered on alternate days, in total fivetimes, starting from the age 4 weeks), intraperitoneal or oral dosing ofglutathione monoethyl ester (2 mg/0.5 ml/animal, administered onalternate days, in total five times, starting from the age 4 weeks),intraperitoneal or oral dosing of glutathione nitroxybutyl ester (0.5mg/0.5 mil/animal, administered on alternate days, in total five times,starting from the age 4 weeks), intraperitoneal or oral dosing ofglutathione diethyl ester (2 mg/0.5 ml/animal, administered on alternatedays, in total six times, starting from the age 3 weeks), andintraperitoneal dosing of lipoic acid (4 mg/0.5 ml/animal, administeredon alternate days, in total five times, starting from the age 4 weeks).

Example 23

Effects of chemical agents administered to NOD mice aged 3 to 6 weeks ata stage of macrophages at oxidized state and with potential occurrenceof inflammatory cell infiltration in pancreatic islet

NOD mice purchased from Nippon Clair, Co. were naturally mated together.Among the offspring mice, a colony of NOD mice with a high frequency ofthe spontaneous onset of insulin-dependent diabetes mellitus wasestablished. Female NOD mice from the colony were used for the presentexperiment. Chemical agents were orally or intraperitoneallyadministered to the NOD mice aged 3 to 6 weeks three times per week, intotal nine times; and based on the test results of urine glucose,positive or negative, the onset of diabetes mellitus was followed onceper week. Urine glucose was detected, using Uropaper (BM Test Glucose5000, manufactured by Yamanouchi Pharmaceuticals, Co. Ltd.).

The results are shown in Table 5.

TABLE 5 Frequency (%) of onset Frequency (%) of onset Chemical Agents ofdiabetes mellitus *a of diabetes mellitus *b Physiological saline 50 80(NAC—OMe)₂ 70 80 (NAC)₂ 60 80 Oral NAC 30 40 Intraperitoneal NAC 10 20Lentinan 10 20 GSHOEt  0  5 *a. on age 18 weeks *b. on age 22 weeks

Chemical agents were administered intraperitoneally, unless otherwisestated.

The aforementioned results based on the experiments of drug efficacyapparently indicate that reductive macrophage-inducing substancesincluding glutathione precursors such as γ-glutamylcysteine,γ-glutamylcysteine dimethyl ester, and N-acetylcysteine nitroxybutylester, glutathione derivatives such as glutathione monoethyl ester,glutathione nitroxybutyl ester and glutathione diethyl diester, andβ(1-3)-linked glucans such as lentinan can suppress the spontaneousonset of diabetes mellitus in NOD mice of age 3 to 6 weeks at a stage ofmacrophages at oxidized state and with potential occurrence ofinflammatory cell infiltration in pancreatic islet. (NAC)₂ and(NAC-OMe)₂were at a single dose of 20 μg/animal; and NAC and GSHOEt wereat a single dose of 2 μg/animal; and a single dose of lentinan was 0.1mg/kg.

Example 24

Effects of chemical agents administered to NOD mice of age 3 to 6 weeksat a stage of macrophages at oxidized state and with potentialoccurrence of inflammatory cell infiltration in pancreatic islet and toage 9 to 11 weeks

NOD mice purchased from Nippon Clair, Co. were naturally mated together.Among the off spring mice, a colony of NOD mice with a high frequency ofthe spontaneous onset of insulin-dependent diabetes mellitus wasestablished. Female NOD mice from the colony were used for the presentexperiment. Chemical agents were orally or intraperitoneallyadministered to the NOD mice of age 3 to 6 weeks three times per weekand to the NOD mice of age 9 to 11 weeks twice per week, in total 15times; and based on the test results of urine glucose, positive ornegative, the onset of diabetes mellitus was followed once per week.Urine glucose was detected, using Uropaper (BM Test Glucose 5000,manufactured by Yamanouchi Pharmaceuticals, Co. Ltd.).

The results are shown in Table 6.

Frequency (%) of onset of diabetes Chemical agents mellitusPhysiological saline 60 Intraperitoneal (NAC—OMe)₂ (20 μg/h) 70 Oral NAC(2 mg/h) 40 Intraperitoneal γ-glutamylcystein dimethyl ester 10 (1 mg/h)Oral γ-glutamylcysteine dimethyl ester (1 mg/h) 20 Intraperitonealglutathione monoethyl ester (1  0 mg/h) Intraperitoneal glutathionediethyl ester (1 mg/h)  0 Lentinan (0.1 mg/kg) Oral glutathione diethyldiester (5 mg/h)  0

The onset was judged on week 22.

The aforementioned results based on the experiments of drug efficacyapparently indicate that reductive macrophage-inducing substancesincluding glutathione precursors such as γ-glutamylcysteine,γ-glutamylcysteine dimethyl ester, and N-acetylcysteine nitroxybutylester, glutathione derivatives such as glutathione monoethyl ester,glutathione nitroxybutyl ester and glutathione diester and β(1-3)-linkedglucans such as lentinan, can suppress the spontaneous onset ofinflammation of pancreatic islet and that of diabetes mellitus owing tothe suppression of inflammatory cell infiltration into pancreatic islet,when these substances are administered to NOD mice of age 3 to 6 weeksat a stage of macrophages at oxidized state and with potentialoccurrence of inflammatory cell infiltration in pancreatic islet, and toage 9 to 11 weeks at a stage of induction of reductive macrophages.

Example 25

Effects of chemical agents administered to NOD mice aged 9 to 11 weeksat a stage of macrophages at reduced state

NOD mice purchased from Nippon Clair, Co. were naturally mated together.Among the off spring mice, a colony of NOD mice with a high frequency ofthe spontaneous onset of insulin-dependent diabetes mellitus wasestablished. Female NOD mice from the colony were used for the presentexperiment. Chemical agents were orally or intraperitoneallyadministered to the NOD mice aged 9 to 11 weeks three times per week, intotal nine times; and based on the test results of urine glucose,positive or negative, the onset of diabetes mellitus was followed onceper week. Urine glucose was detected, using Uropaper (BM Test Glucose5000, manufactured by Yamanouchi Pharmaceuticals, Co. Ltd.).

The results are shown in Table 7.

TABLE 7 Frequency (%) of onset of diabetes Chemical Agents mellitusPhysiological Saline 67 Intraperitoneal (NAC—OMe)₂ 11 (NAlaC—OMe)₂ 11(NAC)₂ 60 Oral NAC 60 Intraperitoneal γ-Glutamylcysteine dimethyl Ester55 Intraperitoneal glutathione diethyl ester 67 Lentinan 50

The onset was judged on week 22; the doses were the same as in Example24.

The aforementioned results apparently indicate that compoundsrepresented by the structural formula 1, including N,N′-diacetylcystine[(NAC)₂], N,N′-dipropylcystine [(NPC)₂], N,N′-diacetylcystine dimethylester [(NAC-OMe)₂], N,N′-diacetylcystine diisopropyl ester [(NAC-OiPr)₂]and N,N′-di-L-alanylcystine dimethyl ester [(NAlaC-OMe)₂], andnitroxybutyl esters thereof and exerting actions to decrease the contentof reduced glutathione in macrophages, suppress delayed typehypersensitivity reactions, and suppress the production of IFNγ orIL-12, can exert drug efficacy when these compounds are administered tothe NOD mice aged 9 to 11 weeks at a stage of macrophages at reducedstate.

Example 26

Effects of chemical agents in db/db mice

db/db mice purchased from Nippon Clair, Co. were naturally matedtogether. Among the off spring mice, a colony of db/db mice with a highfrequency of the spontaneous onset of insulin-non-dependent diabetesmellitus was established. Male db/db mice from the colony were used forthe present experiment. Chemical agents were intraperitoneallyadministered to the db/db mice aged 4 to 9 weeks three times per week,in total 18 times; and based on the level of blood sugar in the mice atsatiation, the effects of the chemical agents on the improvement of thediseased conditions of insulin-non-dependent diabetes mellitus werefollowed once per week.

The results are shown in Table 8.

TABLE 8 Relative ratio % of blood sugar level Chemical Agents 6 weeks 7weeks 8 weeks Physiological saline 100  100  100  (NAC—OMe)₂ 94 92 93(NAC)₂ 95 100  98 γ-Glutamylcysteine 85 65 60 dimethyl ester Glutathionediethyl ester 82 62 60 Lentinan + glutathione 70 55 50 diethyl ester

The doses were the same as in Example 24.

The aforementioned results based on the experiments of drug efficacyapparently indicate that when administered to the db/db mice at a highfrequency of the spontaneous onset of insulin-non-dependent diabetesmellitus, these reductive macrophage-inducing substances includingglutathione precursors such as γ-glutamylcysteine, γ-glutamylcysteinedimethyl ester, and N-acetylcysteine nitroxybutyl ester, glutathionederivatives such as glutathione monoester, glutathione nitroxybutylester and glutathione diester, and β(1-3)-linked glucans such aslentinan, significantly decrease the blood sugar level and effectivelyameliorate the diseased conditions of insulin-non-dependent diabetesmellitus with the etiology of poor glucose incorporation into muscle andfat cells. The action mechanism is not yet elucidated, but thepharmacological effects may possibly be ascribed to the improvement ofliver function or the inhibitory action against phosphatase.

Obviously, numerous modifications and variations of the presentinvention are possible in light of the above teachings. It is thereforeto be understood that within the scope of the appended claims, theinvention may be practiced otherwise than as specifically describedherein.

U.S. patent application Ser. No. 09/181,881, filed on Oct. 29, 1998, isincorporated herein by reference.

Japanese Patent Application Serial No. 308300, filed on Oct. 29, 1998,is incorporated herein by reference.

Japanese Patent Application Serial No. 9-312727, filed on Oct. 27, 1997,is incorporated herein by reference.

What is claimed is:
 1. A method of treating cachectic condition causedby cancers, diabetes, gastrointestinal inflammatory diseases, chronicrheumatoid arthritis, hepatitis, hepatic cirrhosis, hypersensitiveinterstitial pneumonia, pulmonary fibrosis, or autoimmune inflammatorydiseases, comprising administering to a patient in thereof an effectiveamount of a composition comprising a cystine derivative represented byformula (I):

wherein R¹ and R², independently from each other, represent an alkylgroup; and R³ and R⁴, independently from each other, represent an acylgroup or a peptidyl group, wherein said cystine derivative reduces thecontent of reductive glutathione in macrophages, and suppresses cellularimmune responses in said patient.
 2. The method of claim 1, wherein thesubstance has a intramolecular disulfide bond.
 3. The method of claim 2,wherein the substance is a cystine derivative.
 4. The method of claim 1,wherein R¹ and R², independently from each other, represent an alkylgroup having 1 to 12 carbon atoms.
 5. The method of claim 1, wherein R³and R⁴, independently from each other, represent an acyl group having 1to 12 carbon atoms.
 6. The method of claim 5, wherein R¹ and R²,independently from each other, represent an alkyl group having 1 to 12carbon atoms.
 7. The method of claim 1, wherein R³ and R⁴, independentlyfrom each other, represent an peptidyl group having 1 to 10 amino acidresidues.
 8. The method of claim 7, wherein R¹ and R², independentlyfrom each other, represent an alkyl group having 1 to 12 carbon atoms.9. The method of suppressing immune responses according to claim 1,wherein the substance selectively removes reductive macrophages whichcontain an increased amount of intracellular reductive glutathione. 10.The method of suppressing immune responses according to claim 9, whereinsaid substance is a compound in which a cytotoxic DNA alkylating agentis conjugated with glutathione, or one which shows a cytotoxicity afterbeing incorporated into macrophages as a precursor.